Therapeutic agent composition and method of use, for treatment of mild congnitive impairment, depression, and psychological disorders

ABSTRACT

The present invention generally relates to the use of cyclic Prolyl Glycine (“cyclic PG” or “cPG”) and analogues and mimetics thereof, as neuroprotective agents for the treatment and or prevention of cognitive impairment and neurological disorders including but not limited to cerebral ischemia or cerebral infarction, status epilepticus, perinatal asphyxia, anoxia, and cerebral trauma, as well as to the treatment and prevention of chronic neurodegenerative disorders such as Alzheimer&#39;s disease, Parkinson&#39;s disease, and Dementia with Lewy Bodies. The present invention also generally provides manufacturing methods to prepare of dosage forms. The present invention further generally relates to the use of cyclic Prolyl Glycine and analogues and mimetics thereof, as neuroprotective and neuro-regenerating agents for the treatment and or prevention of depression and other psychological disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is:

-   -   a Continuation of U.S. Utility patent application Ser. No.        16/412,759, filed May 15, 2019, now pending; which claims        benefit of priority to    -   U.S. Provisional Patent Application Ser. No. 62/674,855, filed        May 22, 2018, now expired;    -   U.S. Provisional Patent Application Ser. No. 62/671,485, filed        May 15, 2018, now expired; and    -   U.S. Provisional Patent Application Ser. No. 62/671,466, filed        May 15, 2018, now expired;        each of which is incorporated by reference herein in their        entirety.

Inventor Lloyd Hung Loi Tran reflects a legal name change from Loi H.Tran,

FIELD OF THE INVENTION

The present invention generally relates to novel cyclic dipeptidescompounds structurally related to diketopiperazines and methods fortheir therapeutic use. In particular, this invention relates to theneuroprotection and neurogenesis activity of such compounds.Particularly, the present invention relates to the use of cyclic prolylglycine (“cyclic GP” or “cPG”) and cPG analogues and cPG compounds,pharmacologically effective analogues thereof, and pharmaceuticalcompositions thereof in the treatment and prevention of cognitiveimpairment and related neurodegenerative disorders and psychologicaldisorders. The present invention also generally relates to materials andmethods of regenerating neurons and glial cells or a method of repairingdamaged neurons and glial cells.

BACKGROUND

Mild cognitive impairment (“MCI”) is a syndrome defined as cognitivedecline greater than expected for an individual's age and educationlevel. Mild cognitive impairments are those involving impairments ofmemory and other cognition functions, beyond the age norm but notleading to the characteristic of dementia. The prevalence of MCI variesby age. The prevalence of MCI among different age groups is as follows:6.7% for ages 60-64; 8.4% for ages 65-69, 10.1% for ages 70-74, 14.8%for ages 75-79, and 25.2% for ages 80-84. More than half progress ofpeople with MCI progress to dementia within 5 years. (Petersen R C,Lopez O, Armstrong M J, Getchius T, Ganguli M, Gloss D, Gronseth G S,Marson D, Pringsheim T, Day G S, Sager M, Stevens I, Rae-Grant A(January 2018), “Practice guideline update summary: Mild cognitiveimpairment—Report of the Guideline Development, Dissemination, andImplementation Subcommittee of the American Academy of Neurology”.Neurology, Special article, 90 (3): 1-10.:10.1212/WNL.0000000000004826.https://en.wikipedia.org/wiki/PubMed_Identifier“ \o”PubMed Identifier29282327.)

Dementia is an overall term that describes a group of symptomsassociated with a decline in memory or other thinking skills severeenough to reduce a person's ability to perform everyday activities.Alzheimer's disease accounts for 60 to 80 percent of these types ofcases. Vascular dementia, which occurs after a stroke, is the secondmost common dementia type. But there are many other conditions that cancause symptoms of dementia, including some that are reversible, such asthyroid problems and vitamin deficiencies. While symptoms of dementiacan vary greatly, at least two of the following core mental functionsare to be significantly impaired to be considered dementia: memory,communication and language, ability to focus and pay attention,reasoning and judgment, and visual perception.

There are more than 5.5 million people in the US and 50 million peopleworldwide that have Alzheimer's disease in 2018. (Ref: Alzheimer'sDisease International's World Alzheimer Report 2018) The growth in theprevalence of Alzheimer's disease over the next few decades isanticipated to result in great pressure on the social and health-caresystems of developed and developing economies alike. There is a longunmet need for therapies that halt, substantially slow, slow orotherwise ameliorate the progression, symptoms, or provide comfort orpalliative care of this disease and related diseases.

Ramon y Cajal, a pioneer researcher in the early 20^(th) century wrote:“The functional specialization of the brain imposed on the neurons twogreat lacunae; proliferative inability and irreversibility ofintraprotoplasmatic differentiation. It is for this reason that, oncethe development was ended, the founts of growth and regeneration of theaxons and dendrites dried up irrevocably.” (Ramón y Cajal, Santiago; L.Azoulay (1894). Les nouvelles idées sur la structure du systeme nerveuxchez l'homme et chez les vertébrés' (New ideas on the fine anatomy ofthe nerve centres). This hypothesis, while previously being consideredthe fundamental principle of neuroscience dated back in the late 19^(th)century to the mid-20^(th) century, has now been proved invalid.

In 1966 Altman and Gopal demonstrated the evidence of adult mammalianneurogenesis was found in rodent hippocampus and other region of thebrain. He reported the autoradiographic and histological studies ofpostnatal neurogenesis, in which cell proliferation and migration in theanterior forebrain, with special reference to persisting neurogenesis inthe olfactory bulb. ((Altman J. (1966) and D. Gopal. “Autoradiographicand histological evidence of postnatal hippocampal neurogenesis in rats”Journal of Comparative Neurology—Volume 124, Issue 3, June 1965(https://doi.org/10.1002/cne.901240303)).

In Altman's experiment, the Intact adult mammalian brain,neuroregeneration maintains the function and structure of the centralnervous system (CNS). Thymidine-H3 was injected intraperitoneally into6- and 13-day old rats and they lived afterwards for periods rangingfrom one hour to 60 days. Autoradiographic data obtained from animalssurviving for short periods were used to estimate rates of regional cellproliferation. Animals with longer survival were used to deduce themovements of new cells from germinal sites, through migratory channels,to target areas, and to determine their mode of differentiation. Theformation and differentiation of microneurons goes on during infancy,though in most structures at a declining rate. Cell multiplicationcontinued at a very high rate in the external granular layer of thecerebellar cortex, whence cells migrated to the molecular layer andinternal granular layer.

It has been shown that indeed adult regenerated neurons are integratedInto the existing brain circuitry, and contribute to amelioratingneurological deficits (Nakatomi H. Kiiriu T. Okabe S. Yamamoto S. HatanoO. Kawahara N. Tarnura A. Kirino T. Nakafuku M—Regeneration ofhippocampal pyramidal neurons after ischemic brain injury by recruitmentof endogenous neural progenitors. Cell 110:429-441, 2002.

Interestingly, observations have also shown that neurogenesis isoccurring not only at the level of the olfactory bulb and hippocampus.In this respect it has been suggested by Zhao et al. that this processcan also occur in the adult mouse substantia nigra, opening up a newfield of investigation for the treatment of neurogenerative diseases(Zhao M. Momma S. Delfani K. Carlen M. Cassidy R M. Johansson C B.Brisrnar H. Shupliakov O. Frisen J. Janson A M (2003) Evidence forneurogenesis in the adult mammalian substantia nigra. Proc Natl Axad SciUSA 100:7925-7930)

According to a review paper by Guo-li Ming, since the discovery ofneurogenesis the postnatal rat hippocampus, investigators have nowfirmly established that active neurogenesis from neural progenitorscontinues throughout life in discrete regions of the central nervoussystems (CMS) of all mammals, including hurnans. (Guo-li Ming H. S.(2005). “Adult Neurogenesis in the Mammalian Central Nervous System”.Annu. Rev. Neurosci. 28: 223-250. PMID 16022595)

Significant progress has been made in understanding the developmentalprocess and regulation of adult neurogenesis, including proliferation,fate specification, neuronal maturation, targeting, and synapticintegration of the newborn neurons.

While the exact mechanism that maintains functional neural stem cells(NSCs) in these regions is elusive, NSCs have shown an ability torestore neurons and glia in response to certain pathological conditions.

Depression is a mental health disorder characterized by persistentlydepressed mood or loss of interest in activities, causing significantimpairment in daily life. It Is a very common condition that affectsabout 1 in every 5 individuals in the U.S. Many factors can causedepression, including genes, factors such as stress and brain chemistry.According to John Geddes, professor of epidemiological psychiatry atOxford University, “depression is the single largest contributor toglobal disability that we have—a massive challenge for humankind,” Itaffects around 350 million people worldwide and instances has risenalmost 20% from 2005-2015 (World Health Organization and Center forDisease Control).

In recent years, much research effort has been invested in the study ofmental depression and to methods for its treatment. While a number ofdrugs in the market can ameliorate depression, they all have haveundesirable side effects. It has recently been reported that one of theserious side effects of depression is suicide which has virtually becomea sole cause of death from mental illness.

The present invention provides a method for the treatment of depressionwithout serious side effects. According to this method, cyclic ProlylGlycine and its pharmacologically effective analogues have beendemonstrated as a potential treatment a patient suffering fromdepression.

SUMMARY OF THE INVENTION

One aspect the invention provides cyclic Prolyl Glycine compoundssuitable for the treatment and prevention of disease and injury inanimals and humans. The cyclic PG being selected from the group thatincludes cPG, cPG analogues, cPG peptidomimetics and relating compoundswhich promote or cause the formation of cPG or cPG analogues in vivo.

One example of cPG analogues Is cyclic(glycyl-L-prolylglycyl-L-prolylglycyl-L-prolyl) or being abbreviated ascyclic(tri(Pro-Gly)) or referred herein as c(PG)3.

Another example of the cPG analogues is cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline referred herein as “cGAL.”

Collectively the cPG, c(PG)3, cGAL, and Cyclic Glycy 1-2-Methyl-Proline,and their pharmaceutically acceptable salts, are referred hereincollectively as the “cPG compounds.”

Also, Cyclic Glycyl-2-Methyl-Proline is a compound belong to the cyclicGlycyl-2-Alkyl Proline) group of compounds.

Furthermore, any of the cPG compounds, derivatives thereof, analogsthereof, and the like as disclosed herein or otherwise known in the artcan be provided in the form of a pharmaceutically acceptable salt.

Preferably the cPG compounds are administered in a pharmaceuticallyacceptable composition such as a pharmaceutically acceptable carrier.

More preferably the composition additionally includes a therapeuticamount of a cPG compound in combination with a compound selected fromgrowth factors and associated derivatives (insulin-like growth factor-I(IGF-I), insulin-like growth factor-11 (IGF-II), GPE, transforminggrowth factor-ill, activin, growth hormone, nerve growth factor, growthhormone binding protein, JQF-binding proteins (especially JGFBP-3),basic fibroblast growth factor, acidic fibroblast growth factor, thehst/Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor,androgen-induced growth factor. Additional members of the FGF familyinclude, for example, int-2, fibroblast growth factor homologousfactor-1 (FHF-1) FHF-2 FHF-3 and FHF-4, karatinocyte growth factor 2,glial-activating factor, FGF-10 and FGF-16, ciliary neurotrophic factor,brain derived growth factor, neurotrophin 3, neurotrophin 4, bonemorphogenetic protein 2 (BMP-2), ghal-cell line derived neurotrophicfactor, activity-dependant neurotrophic factor, cytokine leukaemiainhibiting factor, oncostatin M, interleukin), β, α, χ or consensusinterferon, TNF-α; clomethiazole; kynurenic acid, Semax, FK506[tacrolimus], L-threo-1-pheyl-2-decanoylamino-3-morpholino-1-propanol,andrenocorticotropin-(4-9_analogue [ORG2766] and dizolcipine [MK-801],selegiline; glutamate ants such as, NPS15O6, GV1505260, MK-801,GV150526; AMP A ants such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164; anti-inflammatory agentsdirected against the addressin MAdCAM-1 and/or it integrin α4 receptors(α4β1 and α 4β7), such as anti-MAdCAM-11mAb MECA-367 (ATCC accession no.(HB-9478), interferons including interferon beta 1b and interferonalfacon-1.

Preferably the cPG compounds can be used in the treatment or preventionof cell damage or cell death in response to diseases and injuryresulting from septic shock, ischemia, administration of cytokines,overexpression of cytokines, ulcers, gastritis, ulcerative colitis,Crohn's disease, diabetes, rheumatoid arthritis, asthma, Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, cirrhosis,allograft rejection, transplant rejection, encephalomyelitis,meningitis, pancreatitis, peritonitis, vasculitis, lymphocyticchoriomeningitis glomerulonephritis, uveitis, glaucoma, blephantis,chalazion, allergic eye disease, corneal ulcer, keratitis, cataract,retinal disorders, age-related macular degeneration, optic neuritisileitis, inflammation induced by overproduction of inflammatorycytokines, hemorrhagic shock, anaphylactic shock, burn, infectionleading to the overproduction of inflammatory cytokines induced bybacteria, virus, fungus, and parasites, hemodialysis, chronic fatiguesyndrome, stroke, cancers, cardiovascular diseases associated withoverproduction of inflammatory cytokines, heart disease, cardiopulmonarybypass, ischemic/reperfusion injury, ischemic/reperfusion associatedwith overproduction of inflammatory cytokines, toxic shock syndrome,adult respiratory distress syndrome, cachexia, myocarditis, autoimmunedisorders, eczema, psoriasis, heart failure, dermatitis, urticaria,cerebral ischemia, systemic lupus erythematosis, AIDS, AIDS dementia,chronic neurodegenerative disease, chronic pain, priapism, cysticfibrosis, amyotrophic lateral sclerosis, schizophrenia, depression,premenstrual syndrome, anxiety, addiction, migraine, Huntington'sdisease, epilepsy, gastrointestinal motility disorders, obesity,hyperphagia, neuroblastoma, malaria, hematologic cancers, myelofibrosis,lung injury, graft-versus-host disease, head injury, CNS trauma,hepatitis, renal failure, chronic hepatitis C, paraquat poisoning,transplant rejection and preservation, fertility enhancement, bacterialtranslocation, circulatory shock, traumatic shock, hemodialysis,hangover, and combinations of two or more thereof.

Preferably the cPG compounds can be used in the restoration ofmyelination of axons in mammals where myelin depleted due to neuralinjury or disease.

Preferably cPG compound can be used in the restoration of myelinationwhere depletion due to trauma, toxin exposure, asphyxia orhypoxia-ischemia, perinatal hypoxic-ischemic injury, injury to ordisease of the white matter of the CNS, acute brain injury, chronicneurodegenerative disease including multiple sclerosis, anddemyelinating diseases and disorders including acute disseminatedencephalomyelitis, optic neuritis, transverse myelitis, Devic's disease,the leucodystrophies; non-inflammatory Involvement; progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

Preferably the cPG compound can be administered in combination withIGF-1 or an interferon.

Another related aspect the invention relates to a method of treating orpreventing cell damage or cell death in response to injury and diseaseby administering at least one cPG compound.

Preferably, the cPG compound can be administered at between about 1 jigto about 150 mg per kilogram of bodyweight. A suitable dosage foradministration of cPG can be, for example, preferably but not limited tobetween about 0.1 mg to about 100 mg per kilogram of body weight,between about 1 mg to about 100 mg per kilogram of body weight, betweenabout 5 mg and about 70 mg per kilogram of body weight, between about 10mg to about 50 mg per kilogram of body weight, or between about 20 mg toabout 40 mg per kilogram of bodyweight. The dose, route ofadministration, and regime of cPG may be different for differentdiseases, disorders, and conditions. As an example, mild cognitiveimpairment may have a lower dose using the same or different route ofadministration than for Alzheimer's disease.

For example: a typical dosage for patient with mild cognitive impairmentcan be between about 0.2 mg to about 1 mg per day administered orally(for example, taking 1 capsule of 20 mg or 2 capsules per day per doctorprescription). Whereas, a more severe Alzheimer's Disease or severeTraumatic Brain injury patent can be administered intravenously in arange from between about 50 mg to about 300 mg per day. The particularsof the dose, route of administration, and regime for a particulardisease, disorder, or condition can be evaluated in general or for aparticular subject or patient.

A further aspect the invention relates to a method of restoring themyelination of axons in a mammal in need of restored myelination due toneural injury or disease, comprising administering a therapeutic amountof a cPG compound, where a cPG compound comprises cPG, a biologicallyactive cPG analogue such as c(PG)3 and cGAL, a biologically active cPGpeptidomimetic, a compound that increases the concentration of cPG, or acompound that increases the concentration of cPG analogues, effective torestore myelination of axons in a mammal. In one aspect of theinvention, the method of restoring myelination of axons comprisingadministering a therapeutic amount of a cPG compound comprisesstimulation of astrocytes to promote remyelination. In another aspect ofthe invention, the method of restoring myelination of axons comprisingadministering a therapeutic amount of a cPG compound comprisesstimulation of oligodendrocytes to produce myelin.

In yet another aspect of the invention, the method of restoringmyelination of axons to a mammal in need of restored myelination furthercomprises administering a therapeutic amount of a cPG compound incombination with a compound selected from IGF-I or an Interferon. In oneaspect of the invention, the method of restoring myelination of axonscomprising administering a therapeutic amount of a cPG compound incombination with IGF-I or an Interferon to stimulate astrocytes topromote remyelination. In another aspect of the invention, the method ofrestoring myelination of axons comprising administering a therapeuticamount of cPG in combination with IGF-I or an interferon to stimulateoligodendrocytes to produce myelin. In preferred embodiments, theinterferon comprises Interferon beta 1b (Betaseron). In a further mostpreferred embodiment, the interferon comprises consensus interferon(Infergen®, interferon alfacon-1).

In still a further aspect of the invention, the methods to treat orprevent cell damage and death in response to injury and disease,comprises administration of a therapeutic amount of a cPG compound,preferably but not limited to an amount from between about 10 μg toabout 150 mg of cPG per kg of body weight of the mammal. A suitabledosage for administration of cPG can be, for example, preferably but notlimited to between about 0.1 mg to about 100 mg per kilogram of bodyweight, between about 1 mg to about 100 mg per kilogram of body weight,between about 5 mg and about 70 mg per kilogram of body weight, betweenabout 10 mg to about 50 mg per kilogram of body weight, or between about20 mg to about 40 mg per kilogram of bodyweight. The dose, route ofadministration, and regime of cPG may be different for differentdiseases, disorders, and conditions. As an example, mild cognitiveimpairment may have a lower dose using the same or different route ofadministration than for Alzheimer's disease. For example: a typicaldosage for patient with mild cognitive impairment may be between about10 mg to about 50 mg per day administered orally (taking 1 capsule of 20mg or 2 capsules per day per doctor prescription). Whereas, a moresevere Alzheimer's Disease or severe Traumatic Brain injury patent maybe administered intravenously in a range from between about 50 mg toabout 300 mg per day. The particulars of the dose, route ofadministration, and regime for a particular disease, disorder, orcondition can be evaluated in general or for a particular subject orpatient.

In yet another aspect of the invention, the method of restoringmyelination of axons to a mammal in need of restored myelination furthercomprises administering a therapeutic amount of a cPG compound incombination with IGF-1 from about 1 mg to about 10 mg of IGF-I per 1 Kgbody weight of the mammal or an interferon from about 1.0 jig to about10 jig of IGF-I per Kg of body weight of the mammal. In a preferredembodiment, the interferon is interferon beta. A suitable dosage foradministration of cPG can be, for example, preferably but not limited tobetween about 0.1 mg to about 100 mg per kilogram of body weight,between about 1 mg to about 100 mg per kilogram of body weight, betweenabout 5 mg and about 70 mg per kilogram of body weight, between about 10mg to about 50 mg per kilogram of body weight, or between about 20 mg toabout 40 mg per kilogram of bodyweight. The dose, route ofadministration, and regime of cPG may be different for differentdiseases, disorders, and conditions. As an example, mild cognitiveimpairment can have a lower dose using the same or different route ofadministration than for Alzheimer's disease. For example: a typicaldosage for patient with mild cognitive impairment can be between about10 mg to about 40 mg per day administered orally (taking 1 capsule of 20mg or 2 capsules per day per doctor prescription). Whereas, a moresevere Alzheimer's Disease or severe Traumatic Brain injury patent canbe administered intravenously m a range from between about 50 mg toabout 300 mg per day. The particulars of the dose, route ofadministration, and regime for a particular disease, disorder, orcondition can be evaluated in general or for a particular subject orpatient.

In a further preferred embodiment of the methods to treat or preventcell damage and death in response to Injury and disease, comprisingadministration of a cPG compound, the cPG compound is administered tothe mammal through a shunt into a ventricle of the mammal.

In a further preferred embodiment of the methods to treat or preventcell damage and death in response to injury and disease, comprisingadministration of a cPG compound, the cPG compound is administered tothe mammal by peripheral administration.

The present invention provides a method of treatment for stimulatingmature astrocytes to promote myelin production after hypoxic-ischemicinjury including the step of increasing the active concentration of cPGand/or the concentration of analogues of cPG in the CNS of mammals.

Most preferably, it is the effective amount of IGF-I itself that isincreased within the CNS of the mammal. This can be effected by directadministration of a cPG compound such as cPG, c(PG)3 or cGAL or cGMePand indeed this is preferred. However, the administration of compoundsthat indirectly increase the effective amount of IGF-I (for example apro-drug which, within the patient is cleaved to release cPG) is in noway excluded.

The active compound (IGF-I or its analogue or its mimetic) can beadministered alone, or as is preferred, a part of a pharmaceuticalcomposition.

The composition can be administered directly to the CNS. The latterroute of administration can involve, for example, lateralcerebroventricular injection, focal injection or a surgically insertedshunt into the lateral cerebroventricle of the brain of the patient.

Conveniently, the stimulation and promotion of myelin production inoligodendrocytes and the support, stimulation and promotion ofremyelination by mature astrocytes is promoted through theadministration of cPG compounds in the prophylaxis or therapy ofdemyelinating diseases such as multiple sclerosis.

As generally described in U.S. Published patent applicationUS20100247483A1 expressly incorporated herein fully by reference, cyclicProlyl Glycine (“cyclic PG” or “cPG”) has the following structure:

Structure 1: Cyclic Prolyl Glycine

(This structure and compound are also known as NA-831 and thoseterminologies are used interchangeably herein)

The present invention includes novel diketopiperazines that arestructurally related to cPG.

One aspect of this invention provides novel cyclic compounds having thestructural formula and substituents described below.

Structure 2: Cyclic Glycyl-2-Allyl Proline, or Cyclic Glycyl-AlkylProline

(referred to herein as “cGAL”)

Where R can be an Alkyl” which refers to a saturated branched, straightchain or cyclic hydrocarbon radical. Exemplary alkyl groups includemethyl, ethyl, isopropyl, cyclopropyl tert-butyl, cyclopropyImethy 1,hexyl and the like.

Where R can be an Ally, which refer to a group is a substituent with thestructural formula H₂C═CH—CH₂R, where R is the rest of the molecule.

With R is a methyl, an aspect of the present invention that includesCyclic Glycyl-2-Alkyl Proline is(8aS)-Methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, which isreferred to as Cyclic Glycy 1-2-Methyl-Pro lime or cyclicGMeP or cGMeP.

Structure 3: Cyclic G-2MeP

(available for purchase from polypeptide suppliers such as BachemAmericas, Inc. (Torrance, Calif., USA)).

In general, c(PG)3 and cGAL can be prepared by methods such as arealready well-known to persons of ordinary skill in the art of peptideand modified peptide synthesis. See for example, Bodanzsky: Principlesof Peptide Synthesis, Berlin, Neve York: Springer-Verlag 1993. Synthesisof the diketopiperazine compounds of this invention may be bysolution-phase synthesis as discussed in the Examples or via thesolid-phase synthesis method exemplified by Merrifield et ah 1963 J.Amer. Chem. Soc.: 85, 2149-2156. Specific examples of diketopiperazinesynthesis can be found in Fischer, 2003, J. Peptide Science: 9: 9-35 andreferences therein. A person of ordinary skill in the art will have nodifficulty, taking account of that skill and the knowledge available,and of this disclosure, in developing one or more suitable syntheticmethods for compounds of this invention.

In the present application, notably but not limited to this sectionwhere compound names and structures and abbreviations are provided, thevarious compounds can all be used in all aspects of the presentinvention included herein. For example, should cPG be indicted in thespecification, then all other compounds of this section (and theapplication as a whole) that are cPG compounds and related derivativessuch as but not limited to cGAL are included in that and otherdescriptions, notably but not limited to methods of treatment of avariety of conditions described herein.

Structure 4: Cyclic (glycyl-L-prolylglycyl-L-prolylglycyl-L-prolyl)

(one possible structure)

The chemical synthesis of cyclic(glycyl-L-prolylglycyl-L-prolylglycyl-L-prolyl) was carried out aspublished in Israel Journal of Chemistry, Vol. 12, Nos. 1-2, 1974, pp.15-29 “CYCLIC Peptides VII: The Synthesis and Characterization of CyclicPeptides with Repeating Pro-Gly Sequences—by Charles M. Deber and ElkanR. Blout.

Synthesis of Cyclo(glycyl-L-prolyl-glycyl-L-prolyl-glycyl-L-prolyl) Asolution of p-nitrophenyl ester hydrochloride (500 mg) dissolved indimethyl-formamide (DMF) (20 ml, dried over sodium sulfate) was addeddropwise with stirring over 6 hours to 500 ml of reagent-grade pyridine,at room temperature. The bright yellow mixture was constantly stirredover 48 hours at room temperature. Solvents were removed byrotary-evaporator-high vacuum pump system at 45°. The residue was washedwith 20 ml of acetone which dissolved the p-nitrophenol and pyridinehydrochloride, but left the peptidic fraction insoluble. The insolublematerials and acetone were transferred to a flask and allow acetone toevaporate at 45°. The material was then dissolved in a minimum of DMT.The white microcrystalline precipitate was shown to beCyclo(glycyl-L-prolyl-glycyl-L-prolyl-glycyl-L-prolyl) (155 mg, with 28%yield), formed complexed with DMF. Crystallization from methanol-etherof 100 mg of this material gave crystalline cyclo(Pro-Gly)₃ (55 mg) freeof DMF.

Chemical analysis: Calculated for C₂₁H₃₀N₆O₆H₂O: C, 52.49; H, 6.71; N,17.49.

Elemental analysis. found C, 52.60; H, 6.81; N, 17.38.

In still other aspects, present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient orcarrier and a therapeutically effective amount of cyclic GP or itsanalogues with structural formulas given above to treat a disease,disorder, or condition, including but not limited to Alzheimer's diseaseand its related conditions such an impairment of cognitive function.

In further aspects, the present invention provides methods of treatingan animal having a cognitive impairment, comprising administration tothat animal an effective amount of a composition comprising cyclic GP orits analogues. In yet further aspects, the animal to be treated is ahuman.

One aspect of the present invention is generally directed to therapeutictreatments of neurological diseases and injuries. Expressly do not wishto be limited to any mechanisms of action, and merely propose anymechanisms of action, the inventor(s) propose that the present inventioncan in part be based on inducing neurogenesis, in particular, neuralstem cell, or progenitor cell proliferation, in accordance with oneaspect of the present invention, cyclic Prolyl Glycine and it analogues(“cPG compounds”) act as key neurogenesis modulating agents thatfacilitate and induce proliferation and/or differentiation in neuralcells.

“Neurogenesis” is defined herein as proliferation, differentiation,migration, or survival of a neural cell in vivo or in vitro. In apreferred aspect of the present invention, the neural cell can be anadult, fetal, or embryonic neural stem cell or progenitor cell.Neurogenesis also refers to a net increase in cell number or a netincrease in ceil survival. As used herein, “NSC” would include, at leastall brain stem ceils, all brain progenitor cells, and all brainprecursor cells.

It has been previously shown that increased levels of cAMP and/or Ca²⁺elicit the proliferation of adult neural stem cells. In some cases, thisinduction follows the activation of G-protein coupled receptors (GPCRs).Increasing intracellular cAMP and/or Ca²⁺ levels through GPCR ligandscan induce the increase of proliferation of adult neural stem cells.

G-protein-coupled receptors (GPCRs), also known G protein-linkedreceptors (GPLR), constitute a large protein family of receptors thatdetect molecules outside the cell and activate internal signaltransduction pathways and, ultimately, cellular responses.

The ligands that bind and activate these receptors includelight-sensitive compounds, odors, pheromones, hormones, andneurotransmitters, and vary in size from small molecules to peptides tolarge proteins. GPCRs in the mammalian brain bind several differentneurotransmitters, including serotonin, dopamine, GABA, and glutamate. Gprotein-coupled receptors are involved in many diseases, and are alsothe target of approximately 34% of all modern medicinal drugs.

One aspect of the present invention includes, and not being limited fayany proposed mechanisms, that cPG and its analogues can act as aneurogenesis modulating agents that modulate intracellular levels ofcAMP and/or Ca²⁺. cPG has been shown chemically and biologically to becapable of increasing cAMP (e.g., by increasing synthesis or decreasingbreakdown) and/or Ca²⁺ (e.g., by increasing influx or decreasingefflux).

One aspect of the present invention describes a new method for promotingregeneration of damaged nerve tissue, comprising administering aneffective amount of cyclo Prolyl Glycine (cPG), and its analogues, whichcan reduce the rate of growth of glial cells to facilitate the growth ofnerve tissue.

Neurons are closely surrounded by glial cells or astrocytes. One of thedifficulties in achieving regeneration of neurons after they have beendamaged or severed is that the glial cells proliferate and form a banderto the regenerating neurons. The result is that the further movement ofthe neurons toward anticipated attachment sites is blocked andregeneration of structure and function ceases. It has been observed thatformation of astrocytic and connective tissue scars and progressivenecrosis have negative impact on the regeneration of neuronal functions.

Accordingly, one aspect of the present invention is a method forpromoting regeneration of damaged nerve tissue in a mammal (such as ahuman), comprising administering an effective amount of cPG compounds(cPG and its analogues) to the damage site.

Some objectives of the experiments provided herein is to provideenablement for a method of regenerating neurons and glial cells or amethod of repairing damaged neurons and glial cells as claimed.

One aspect of the invention includes, that cyclic Prolyl Glycine (cPG)and its pharmaceutically active analogues act as a neuronal modulatingagents in order to treat depression and other psychological disorders.The N-methyl-D-aspartate receptor (“NMDA receptor”), is a glutamatereceptor and ion channel protein found in nerve cells. The NMDA receptoris one of three types of ionotropic glutamate receptors, the othersbeing the AMPA and kainate receptors. The NMDA receptor is activatedwhen glutamate and glycine bind to it, and when activated it allowspositively charged ions to flow through the cell membrane [Furukawa,Hiroyasu; Singh, Satinder K; Mancussol, Romina; Gouaux, Eric (November2005). “Subunit arrangement and function in NMDA. receptors”. Nature.438 (7065): 185-92. doi:10.1038/nature04089. PMID 16281028.].

The NMDA receptor channels play an important role in synaptic plasticityand synapse formation underlying memory, learning and formation ofneural networks during development in the central nervous system (CNS).Over activation of the receptor, causing excessive influx of Ca²⁺ canlead to excitotoxicity which is implied to be involved in someneurodegenerative disorders. Blocking of NMDA receptors could therefore,in theory, be useful in treating such diseases

The NMDA receptor is an ion channel protein receptor that is activatedwhen glycine and glutamate bind to it. The receptor is a heteromericcomplex that interacts with multiple intracellular proteins by threedifferent subunits: NR1, NR2 and NR3. NR1 has eight different subunitsgenerated by alternative splicing from a single gene. There are fourdifferent NR2 subunits (A-D), and NR3A and NR3B subunits have beenreported. Six separate genes encode for NR2 and NR3. [Loftis J. M.,Janowsky A. (2003). “The N-methyl-D-aspartate receptor subunit NR2B:localization, functional properties, regulation, and clinicalimplications”. Pharmacol Ther. 97 (1): 55-85, doi:10.1016/s0163-7258(02)00302-9.

Agonists or allosteric modulators of NMDA receptors, in particular NR2Bsubunit-containing channels, have been investigated as therapeuticagents for the treatment of major depressive disorder (G. Sanacora,2008, Nature Rev. Drug Disc. 7: 426-437). The NR2B subunit has beeninvolved in modulating activity such as learning, memory, processing andfeeding behaviors, as well as being implicated in number of humanderangement. The basic structure and functions associated with the NMDAreceptor can be attributed to the NR2B subunit.

An allosteric, non-competitive binding site has also been identified inthe N-terminal domain of NR2B. The NR2 subunit acts as the binding sitefor glutamate, one of the predominant excitatory neurotransmitterreceptors in the mammalian brain. [Yoshimura Y, Ohmura T, Komatsu Y(July 2003). “Two forms of synaptic plasticity with distinct dependenceon age, experience, and NMDA receptor subtype in rat visual cortex”. TheJournal of Neuroscience. 23 (16): 6557-66. PMID 12878697].

NR2B has been associated with age- and visual-experience-dependentplasticity in the neocortex of rats, where an increased NR2B/NR2A ratiocorrelates directly with the stronger excitatory DTP in young animals.This is thought to contribute to experience-dependent refinement ofdeveloping cortical circuits.

The role of NR2B subunit of the NMDA receptor has been demonstrated inthe action of different antidepressant agents. [Poleszak E, Wlaź P,Szewczyk B, Wlaź A, Kasperek R, Wróbel A, Nowak G (2011) A complexinteraction between glycine/NMDA receptors andserotonergic/noradrenergic antidepressants in the forced swim test inmice. J Neural Transm 118:1535-1546].

G-protein-coupled receptors (GPCRs), also known G protein-linkedreceptors (GPLR), constitute a large protein family of receptors thatdetect molecules outside the cell and activate internal signaltransduction pathways and, ultimately, cellular responses.

GPCRs in the mammalian brain bind several different neurotransmitters,including serotonin, dopamine, GABA, and glutamate. G protein-coupledreceptors are involved in many diseases and are also the target ofapproximately 34% of all modern medicinal drugs.

One aspect of the invention includes, that cyclic Prolyl Glycine (cPG)and its pharmaceutically active analogues act as a neuronal modulatingagents in order to treat depression and other psychological disorders.One possible mechanism, though expressly not being limited to anymechanism, is the modulation of intracellular levels of cAMP and/orCa²⁺. Herein, cPG has been shown chemically and biologically to becapable of increasing cAMP (e.g., by increasing synthesis or decreasingbreakdown) and/or Ca²⁺ (e.g., by increasing influx or decreasingefflux).

In addition, cyclic Prolyl Glycine and its pharmaceutically activeanalogues have been shown to selectively bind the N-terminal domain ofNR2B, which might sustain an antidepressant response in human.

The present invention provides technical advantages of cyclic ProlylGlycine (“cPG”) and its pharmaceutically active analogues, together isknown as cPG compounds, which are ligands for the NR2B receptor and canbe useful for the treatment of various disorders of the central nervoussystem. In addition, the cPG compounds provide advantages forpharmaceutical uses, for example, with regard to one or more of theirmechanism of action, binding, inhibition efficacy, target selectivity,solubility, safety profiles, or bioavailability.

In carrying out the method of the invention, depressed patients aregiven the combination of substances at pharmaceutically effective dosagelevels using appropriate routes of administration and regime. Thesubstances can be administered m the form of a single dosage unit inwhich the active ingredients are combined with a suitable carrier; orthey may be given in separate dosage units in which the active materialsare individually combined with a suitable carrier. When administeredseparately, the administration may be simultaneous or at selected timeintervals.

The administration is preferably orally and the carrier or carriers areselected with this in mind. While this is the case, other modes ofadministration of both substances as well as mixed modes with theindividual materials is considered part of the present invention.

The dosage levels of the materials will vary with, the particularmaterial being used and the severity of the condition of the patientbeing treated. The cyclic Prolyl Glycine (cPG) is used in amountsranging from about 0.1 mg to about 10 mg per kg of body weight. It isrecommended to administer orally with a dose of about 20 mg to about 80mg per day and can be up to about 100 mg per day for some severe casesper physician's prescription order.

The pharmaceutical compositions of the present invention are prepared byutilizing the active ingredients in association with the pharmaceuticalcarriers conventionally employed with such materials. The compositionsof the present invention are in general contemplated for administrationorally to achieve an antidepressant effect. This may be in any of thedosage forms such as tablets, capsules, powders, suspensions, solutions,syrups and the like, including sustained release preparations. The termdosage form as used in this specification and the claims refer tophysically discrete units to be administered in single or multipledosage, each unit containing a predetermined quantity of active materialin association with the required diluent, carrier or vehicle. Thequantity of active material is that calculated to produce the desiredtherapeutic effect upon administration of one or more of such units.

Powders are prepared by comminuting the active substances to a suitablycontainer size and mixing with a similarly comminuted diluentpharmaceutical carrier such as an edible carbohydrate material as forexample, starch. Sweetening, flavoring, preservative, dispersing andcoloring agents can also be present.

Capsules are made by preparing a powder mixture as described above andfilling formed gelatin sheaths. A lubricant such as talc, magnesiumstearate and calcium stearate can be added to the powder mixture as anadjuvant before the filing operation; a glidant such as colloidal silicamay be added to improve flowing properties; a disintegrating orsolubilizing agent may be added to improve the availability of themedicament when the capsule is ingested.

Tablets are made by preparing a powder mixture, granulating or slugging,adding a lubricant and disintegrant and pressing into tablets. A powdermixture is prepared by mixing the active substance, suitably comminuted,with a diluent or base such as starch, sucrose, kaolin, dicalciumphosphate and the like. The powder mixture can be granulated by wettingwith a binder such as syrup, starch paste, acacia mucilage or solutionsof cellulosic or polymeric materials and forcing through a screen. As analternative to granulating, the powder mixture can be run through thetablet machine and the resulting imperfectly formed slugs broken intogranules. The granules can be lubricated to prevent sticking to thetablet forming dies by means of the addition of stearic acid, a stearatesalt talc or mineral oil. The lubricated mixture is then compressed intotablets.

The medication can also be combined with free-flowing inert carriers andcompressed into tablets directly without going through the granulatingor slugging steps. A protective coating consisting of a seating coat ofshellac, a coating of sugar or polymeric material and a polish coatingof wax can be provided. Dyestuffs can be added to these coatings todistinguish different unit dosages.

Oral liquid formulation such as syrups and elixirs can be prepared inunit dosage form so that a given quantity, e.g., a teaspoonful, containsa predetermined amount of the compound. Syrups can be prepared bydissolving the compound in a suitably flavored aqueous sucrose solutionwhile elixirs are prepared through the use of a nontoxic alcoholicvehicle. Suspensions can be formulated by dispersing the medication in anontoxic vehicle in which it is insoluble.

One important embodiment of the present invention, particularly forpreparing solid pharmaceutical formulations, is the pharmaceuticallyacceptable nontoxic acid addition salts of the active drugs. Suchpharmaceutically acceptable nontoxic salts include those derived fromboth organic and inorganic acids such as, without limitation,hydrochloric, hydrobromic, sulfuric, phosphoric, methanesufonic, acetic,lactic, succinic, malic, maleic, aconitic, phthalic, tartaric, embonic,enanthic and the like acids.

While the present invention contemplates, primarily, oraladministration, other modes are certainly not excluded. Ampules forparenteral application can be prepared and preferably contain watersoluble salts of the active substances and possible buffer substances inaqueous solution.

In liquid compositions, whether designed for oral or parenteraladministration in which the active substances are combined, care must betaken to insure stability of the active materials.

In cases where the active materials are to be administered separately,individual compositions are prepared in the manner indicated above.These individual compositions can then be administered as such orcombined into a single-dosage unit while maintaining the separateidentity, as for example in a multilayer tablet or single capsulecontaining both components in a plurality of discrete particles.

BRIEF DESCRIPTION OF DRAWINGS

A better understanding of the invention will be gained from reference tothe following examples and drawings wherein:

FIG. 1 illustrates the proposed metabolism pathway of cis-GPE to cyclicProlyl Glycine and glutamic acid.

FIG. 2 illustrates the proposed mechanism by which cyclic Prolyl Glycinemay act to bind metal ions.

FIG. 3 illustrates in graphic form Glutamate toxicity in cerebellarmicroexplants (P4) and rescue effect by cyclic GP.

FIG. 4 illustrates in graphic form prevention of glutamate toxicity bycyclic GP monitored within P4-cerebellar microexplants.

FIG. 5 illustrates in graphic form effects of cPG on functional recoveryafter 6-OHDA lesion.

FIG. 6 illustrates the time to reach platform. Morris Water Maze (MWM)Model of Learning and Memory Used to Assess Effects of cyclic ProlylGlycine on Cognitive Function.

FIG. 7 Graph showing the impact of BrdU+Cells/300 micrometer at subventricular zone for cPG, cGMeP and c(PG)3 drug solutions.

FIG. 8 illustrates the impact of BrdU+Cells/300 micrometer at dentategyrus for cPG, c(PG)3 cGMeP and and drug solutions.

FIG. 9 illustrates the improvement in the Ability to Concentrate andCount.

FIG. 10 illustrates the improvement in Short Term Memory.

FIG. 11 illustrates the improvement in Long Term Memory.

FIG. 12 illustrates the Impact on Orientation.

FIG. 13 illustrates illustrates the improvement in Daily Activities andSelf-Care.

FIG. 14 illustrates illustrates the Impact on Emotion.

FIG. 15 illustrates illustrates the Impact of Cognitive Capacity.

The present invention Is described with reference to specificembodiments thereof. Other aspects of this invention can be appreciatedwith reference to the drawings. Drawings have been provided in the abovetext and attached drawings as well, and are further provided below andtheir description provided at that location.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are given by way of illustration only and shallnot be taken as limiting the scope of the invention.

It has been surprisingly discovered that the process of the metabolismof IGF 1 to the tripeptide GPE and des IGF is only a part of theprocess.

The cis-isomer of the GPE can further break down to form a cyclic ProlylGlycine and glutamic acid. This is shown in FIG. 1.

The cyclic PG structure is sufficiently small to allow it to cross theblood-brain barrier.

In addition, as shown in FIG. 2 the structure of the molecule is suchthat it is able to provide ligands for binding metal ions such as Mg²⁺,Ca²⁺, Co²⁺ and the like and as such can act as a chelating agent.

The possible role of cPG as an agent is further supported by thecompanion break down product, glutamic acid.

Glutamic acid is known to be associated with brain disease. (Johnston,G. A. R. in Roberts P. J. et al Editors, Glutamate: Transmitter in theCentral Nervous System, John Wiley & Sons, 1981, pp. 77-87).

As used herein, a cPG compound is a compound with biological activitysimilar or identical to the biological activity of cPG; cPG compoundscomprise cPG, biologically active cPG analogues, biologically active cPGmimetics, and compounds that increase the concentration of cPG and cPGanalogues in a mammal. cPG compounds include cPG molecules such astruncated portions of IGF-I compounds as well as other chemical andbiological analogues and mimetics.

As used herein, “cPG analogue” is any analogue of cPG, naturallyoccurring analogue of cPG, or any variants thereof, which are capable ofeffectively binding to mGluR receptors in the CNS and of promoting anequivalent neuroprotective effect on CNS nerve cells. Examples of cPGanalogues are c(PG)3 and cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or Cyclic Glycyl-2-Methyl-Proline.

The term “cPG molecules” includes peptide fragments and truncatedportions of longer IGF-I compounds as well as other chemical andbiological analogues and mimetics. cPG compounds can be used in thetreatment of mammals, suffering from neutral injury or disease. Inparticular the cPG compounds can be used to treat human patients,suffering from neural injury or disease. Still more generally, thecompositions and methods of the invention find use in the treatment ofmammals, such as human patients, suffering from nerve damage orpotential apoptotic and/or necrotic cell death, doe to injuries anddiseases such as septic shock, ischemia, administration of cytokines,overexpression of cytokines, ulcers, gastritis, ulcerative colitis,Crohn's disease, diabetes, rheumatoid arthritis, asthma, Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, cirrhosis,allograft rejection, transplant rejection, encephalomyelitis,meningitis, pancreatitis, peritonitis, vasculitis, lymphocyticchoriomeningitis, glomerulonephritis, uveitis, glaucoma, blepharitis,chalazion, allergic eye disease, corneal ulcer, keratitis, cataract,retinal disorders, age-related macular degeneration, optic neuritisileitis, inflammation induced by overproduction of inflammatorycytokines, hemorrhagic shock, anaphylactic shock, burn, infectionleading to the overproduction of inflammatory cytokines Induced bybacteria, virus, fungus, and parasites, hemodialysis, chronic fatiguesyndrome, stroke, cancers, cardiovascular diseases associated withoverproduction of inflammatory cytokines, heart disease, cardiopulmonarybypass, ischemic/reperfusion injury, ischemic/reperfusion associatedwith overproduction of Inflammatory cytokines, toxic shock syndrome,adult respiratory distress syndrome, cachexia, myocarditis, autoimmunedisorders, eczema, psoriasis, heart failure, dermatitis, urticaria,cerebral ischemia, systemic lupus erythematosis, AIDS, AIDS dementia,chronic neurodegenerative disease, chronic pain, priapism, cysticfibrosis, amyotrophic lateral sclerosis, schizophrenia, depression,premenstrual syndrome, anxiety, addiction, migraine, Huntington'sdisease, epilepsy, gastrointestinal motility disorders, obesity,hyperphagia, neuroblastoma, malaria, hematologic cancers, myelofibrosis,lung injury, graft-versus-host disease, head injury, CNS trauma,hepatitis, renal failure, chronic hepatitis C, paraquat poisoning,transplant rejection and preservation, fertility enhancement, bacterialtranslocation, circulatory shock, traumatic shock, hemodialysis,hangover, and combinations of two or more thereof.

In addition, cPG and its analogues, such as but not limited to c(PG)3and cGMeP can be used to treat mammals suffering from white matterInsult as the result of acute brain injury, such as perinatalhypoxic-ischemic injury; or from chronic neural injury orneurodegenerative disease, such as multiple sclerosis, or from otherdemyelinating diseases and disorders Including inflammatory involvement,such as acute disseminated encephalomyelitis, optic neuritis, transversemyelitis, Devic's disease, the leucodystrophies; non-inflammatoryinvolvement; progressive multifocal leukoencephalopathy, central pontinemyelinolysis. Patients suffering from such diseases or Injuries willbenefit greatly by a treatment protocol able to initiate remyelination.

The present invention has application in the induction of myelinproduction following insult in the form of trauma, toxin exposure,asphyxia or hypoxia-ischemia, and has application in the treatment orprevention of apoptosis in response to injury or disease in the form ofcancers, viral infections, autoimmune diseases, neurological diseasesand injuries and cardiovascular diseases.

Treatment with cPG or its analogues, including but not limited to c(PG)3and cGAL can be given before (as well as alter) an injury—as for examplebefore elective surgery. Examples of relevant elective proceduresinclude neural surgery, in which retraction of lobes of the brain canlead to cerebral oedema, or heart operations, such as valve replacement,in which inevitable small emboli are said to lead to detectableimpairment of brain function in some 75% of cases.

Pharmacology and Utility

cPG can act as an anti-necrotic and anti-apoptotic in a process of celldeath. Its anti-apoptotic and anti-necrotic activity in vivo can bemeasured by cell counts. cPG can also be measured in vitro. (GudashevaT. A. et al. FEBS Letters, Vol. 391, Issues 1-2, 5 Aug. 1996, pp.149-152). CNS damage may for example be measured clinically by thedegree of permanent neurological deficit cognitive function, and/orpropensity to seizure disorders. (Rakic L. J et al, in Rakic L. J et alPeptide and Amino Acid Transport Mechanisms in The Central NervousSystem, 1988, The MacMillan Press Ltd. (London) pp. 167-181).

Pharmaceutical Compositions and Administration

cPG itself as part of the present invention can be used to prevent ortreat cell damage and programmed death and the induction of myelinproduction. Usually this is effected through the direct administrationof cGP to the patient. If desired, a combination of the cPG compoundsand its analogues can be administered in a pharmaceutically acceptablecomposition.

Those skilled in the art will appreciate there is no intention on thepart of the applicants to exclude administration of other forms of cPGand Its analogues. By way of example, the effective amount of cPG in theCNS can be increased by administration of a pro-drug from of cPG, whichcomprises cPG and a carrier, cPG and the carrier being joined by alinkage which is susceptible to cleavage or digested within the patient.Any suitable linkage can be employed which will be cleaved or digestedto release cPG following administration.

In addition, it Is envisaged cPG levels may be increased through animplant that includes a cell line capable of expressing cPG in an activefrom within the CNS of the patient.

Pro-drugs of cPG and its analogues can also be administered. In thatinstance, the pro-drug is metabolized or otherwise altered within thesubject to form cPG. cPG and its analogues, such as but not limited toc(PG)3 and cyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Prolineor Cyclic Glycyl-2-Methyl-Prolime can be administered as part of amedicament or pharmaceutical preparation. This can involve combining cPGwith any pharmaceutically appropriate carrier, adjuvant or excipient.The selection of the carrier, adjuvant or excipient will of courseusually be dependent upon the route of administration to be employed.

The administration route can vary widely and be any appropriate route ofadministration. An advantage of cPG is that it can be administeredperipherally. This means it need not be administered directly to the CNSof the patient in order to have effect in the CNS.

Any peripheral route known in the art can be employed. These can includebut are not limited to parenteral routes for example injection into theperipheral circulation, subcutaneous, intraorbital, ophthalmic,intraspinal, intracisternal, topical, infusion (using e.g., controlledrelease devices or minipumps such as osmotic pumps or skin patches),implant, aerosol, inhalation, scarification, intraperitoneal,intracapsular, intramuscular, intranasal, oral, buccal, pulmonary,rectal or vaginal. The compositions can be formulated for parenteraladministration to humans or other mammals in therapeutically effectiveamounts (e.g., amounts which eliminate or reduce the patient'spathological condition) to provide therapy for the neurological diseasesdescribed above.

Two of the preferred administration routes will be by subcutaneousinjection (such as but not limited to, dissolved in 0.9% sodiumchloride) or orally (in a capsule).

It will also be appreciated that on occasion it may desirable todirectly administer cPG compounds to the CMS of the patient. Again, thiscan be achieved by any appropriate direct administration route. Examplesinclude administration by lateral cerebroventricular injection orthrough a surgically inserted shunt into the lateral cerebroventricle ofthe brain of the patient.

The calculation of the effective amount of cPG compounds to beadministered is within the skill of one of ordinary skill in the art,and will be routine to those persons skilled in the art. Needless tosay, the final amount to be administered will be dependent upon theroute of administration and upon the nature of the neurological disorderor condition that is to be treated. Preferably the cPG compound will beadministered at between about 1 μg to 100 mg of cPG compound per perkilogram of body weight where the dose is administered centrally. Asuitable dosage for administration of cPG may be, for example, atbetween about 0.1 mg to about 10 mg per per kilogram of body weight, orat between about 1 mg to about 5 mg per per kilogram body weight.

For inclusion in a medicament, cPG compounds can be obtained from asuitable commercial source such as Bachem AG of Bubendorf, Switzerland.Alternatively, cPG can be directly synthesized by conventional methodssuch as the stepwise solid phase synthesis method of Merrifield et al.1963 J. Amer. Chem. Soc.: 85, 2149-2156. Alternatively synthesis caninvolve in the use of commercially available peptide synthesizers suchas the Applied Biosystems model 430A.

cGAL can be prepared by methods such as are well-known to those ofordinary skill in the art of the synthesis of peptides and analogues.Example: “Principles of Peptide Synthesis” by Bodanzsky, published bySpringer-Verlag 1993.

c(PG)3 can be prepared by the method published in the Israel Journal ofChemistry, Vol. 12, Nos. 1-2, 1974, pp, 15-29 “CYCLIC Peptides VII: TheSynthesis and Characterization of Cyclic Peptides with Repeating Pro-GlySequences—by Charles M. Deber and Elkan R. Blout.pu.

As a general proposition, the total pharmaceutically effective amount ofthe cPG compound administered parenterally per dose will be in a rangethat can be measured by a dose response curve. One can administerincreasing amounts of the cPG compound to the patient and check theserum levels of the patient for cPG. The amount of cPG compound to beemployed can be calculated on a molar basis based on these serum levelsof cPG.

Specifically, one method for determining appropriate dosing of thecompound entails measuring cPG levels in a biological fluid such as abody or blood fluid. Measuring such levels can be done by any means,including RIA and ELISA. After measuring cPG levels, the fluid iscontacted with the compound using single or multiple doses. Alter thiscontacting step, the cPG levels are re-measured in the fluid. If thefluid cPG levels have fallen by an amount sufficient to produce thedesired efficacy for which the molecule is to be administered, then thedose of the molecule can be adjusted to produce maximal efficacy. Thismethod can be carried out in vitro or in vivo. Preferably, this methodis carried out in vivo, i.e., after the fluid is extracted from a mammaland the cPG levels measured, the compound herein is administered to themammal using single or multiple doses (that is, the contacting step isachieved by administration to a mammal) and then the cPG levels areremeasured from fluid extracted from the mammal.

The compound may also be suitably administered by a sustained-releasesystem. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et ah, 1983), poly(2-hydroxyethylmethacrylate) (Langer et al, 1981), ethylene vinyl acetate (Langer etal., supra), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also include a liposomally entrappedcompound. Liposomes containing the compound are prepared by methodsknown per se: DE Patent 3,218,121; Epstein et al, 1985; Hwang et ah,1980; EP Patent 52,322; EP Patent 36,676; EP Patent 88,046; EP Patent143,949; EP Patent 142,641; Japanese Pat. Appln. 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomesare of the small (from or about 200 to 800 Angstroms) unilamellar typein which the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the mostefficacious therapy.

PEGylated peptides having a longer life can also be employed, based on,e.g., the conjugate technology described in WO 95/32003, published Nov.30, 1995.

If parenteral administration is preferred, the compound is formulatedgenerally by mixing each at the desired concentration. In a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically, or parenterally, acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the compound withliquid carriers or finely divided solid carriers or both. Then, ifnecessary, the product is shaped Into the desired formulation.Preferably the carrier is a parenteral carrier, more preferably asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline, Ringer's solution, abuffered solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate may also be used.

The carrier may additionally contain additives such as substances thatenhance isotonicity and chemical stability. Such materials are non-toxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, succinate, acetic acid, and otherorganic acids or their salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) polypeptides, e.g.,polyarginine or tripeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;glycine; amino acids such as glutamic acid, aspartic acid, histidine, orarginine; monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, glucose, mannose, trehalose, ordextrin; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; counter-ions such as sodium; non-ionic surfactants such aspolysorbates, poloxamers, or polyethylene glycol (PEG); and/or neutralsalts, e.g., NaCl, KCl, MgCl₂ CaCl₂ etc.

The cPG compound is typically formulated in such vehicles at a pH ofbetween about 5.5 to about 8.0. Typical adjuvants which may beincorporated into tablets, capsules, and the like are a binder such asacacia, corn starch, or gelatin; an excipient such as microcrystallinecellulose; a disintegrating agent like corn starch or alginic acid; alubricant such as magnesium stearate; a sweetening agent such as sucroseor lactose; a flavoring agent such as peppermint, wintergreen, orcherry. When the dosage form is a capsule, in addition to the abovematerials, it may also contain a liquid carrier such as a fatty oil.Other materials of various types may be used as coatings or as modifiersof the physical form of the dosage unit. A syrup or elixir may containthe active compound, a sweetener such as sucrose, preservatives likepropyl paraben, a coloring agent, and a flavoring agent such as cherry.Sterile compositions for injection can be formulated according toconventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as water ornaturally occurring vegetable oil like sesame, peanut, or cottonseed oilor a synthetic fatty vehicle like ethyl oleate or the like may bedesired. Buffers, preservatives, antioxidants, and the like can beincorporated according to accepted pharmaceutical practice.

The compound to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The compound ordinarily will be stored in unit or multi-dose containers,for example, sealed glass ampules or vials, as an aqueous solution or asa lyophilized formulation for reconstitution. As an example of alyophilized formulation, IQ-mL vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous solution of compound, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized compound using bacteriostaticWater-for-Injection.

Combination therapy with the cPG compound herein and one or more otherappropriate reagents that increase total cPG in the blood or enhance theeffect of the cPG is also contemplated. These reagents generally allowthe cPG compound herein to release the generated cPG.

In addition, one aspect of the present invention includes using genetherapy for treating a mammal, using nucleic acid encoding the cPGcompound, if it is a peptide. Generally, gene therapy is used toincrease (or overexpress) cPG levels in the mammal. Nucleic acids, winchencode the cPG peptide can be used for this purpose. Once the amino acidsequence is known, one can generate several nucleic acid molecules usingthe degeneracy of the genetic code, and select which to use for genetherapy.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells for purposes of genetherapy: in vivo and ex vivo. For in vivo delivery, the nucleic acid isinjected directly into the patient, usually at the site where the cPGcompound is required. For ex vivo treatment, the patient's cells areremoved, the nucleic acid is introduced into these isolated cells, andthe modified cells are administered to the patient either directly or,for example, encapsulated within porous membranes which are implantedinto the patient. See, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it Is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,1987; Wagner et al., 1990). For review of the currently known genemarking and gene therapy protocols, see Anderson 1992. See also WO93/25673 and the references cited therein.

Kits are also contemplated for this invention. A typical kit wouldcomprise a container, preferably a vial, for the cPG compoundformulation comprising cPG compound in a pharmaceutically acceptablebuffer and instructions, such as a product insert or label, directingthe user to utilize the pharmaceutical formulation.

Certain aspects of the present invention include the use of cPG intreatment of cognitive impairment associated with aging withneurodegenerative conditions or in situations in which cognitiveimpairment is found with no apparent neurodegeneration.

Such other agents can be selected from the non-limiting group of, forexample, growth factors and associated derivatives, e.g., insulin-likegrowth factor-1 (IGF-I), insulin-like growth factor-II (IGF-II), growthhormone, nerve growth factor, growth hormone binding protein, and/orIGF-binding proteins.

Therapeutic Applications

Compositions and methods of the present invention find use in thetreatment of animals, such as human patients, suffering from cognitiveimpairment. Still more generally, the compositions and methods of theinvention find use in the treatment of mammals, such as but not limitedhuman patients and subjects, suffering from memory impairment, mildcognitive impairment, dementia, including dementia including dementiasresulting from cerebral atrophy associated with Alzheimer's disease,Lewy-bodies disease, frontotemporal lobar degeneration, vasculardementia, head trauma; Huntington's disease, Parkinson's disease, orDown's syndrome.

Pharmaceutical Compositions and Administration

Cyclic PG compounds can be administered as part of a medicament orpharmaceutical preparation. This can involve combining a compound of theinvention with any pharmaceutically appropriate carrier, adjuvant orexcipient. The selection of the carrier, adjuvant or excipient will ofcourse usually be dependent upon the route of administration to beemployed.

In general, compounds of the present invention will be administered intherapeutically effective amounts by any of the usual modes known in theart, either singly or in combination with other conventional therapeuticagents for the disease being treated. A therapeutically effective amountmay vary depending on the disease or injury, its severity, the age andrelative health of the animal being treated, the potency of thecompound(s), and other factors. Therapeutically effective amounts ofcyclic Prolyl Glycine can range from 0.01 to 10 milligrams per kilogrammass of the animal, with lower doses such as 0.01 to 0.1 mg/kg beingappropriate for administration through the cerebrospinal fluid, such asby intracerebroventricular administration, and higher doses such as 0.1to 10 mg/kg being appropriate for administration by methods such asoral, systemic (e.g. transdermal), or parenteral (e.g. intravenous)administration. A person of ordinary skill in the art will be ablewithout undue experimentation, having regard to that skill and thisdisclosure, to determine a therapeutically effective amount of acompound of this invention for a given disease or injury.

Cyclic Prolyl Glycine and cPG compound can be administered orally orperipherally via any peripheral route known in the art. These caninclude but are not limited to parenteral routes for example injectioninto the peripheral circulation, subcutaneous, intraorbital, ophthalmic,intraspinal, intracisternal, topical, intravenous infusion, aerosol,inhalation, scarification, intraperitoneal, intracapsular,intramuscular, intranasal, buccal, transdermal, pulmonary, rectal orvaginal.

For the convenience to the patients, the cyclic Prolyl Glycine and cPGcompounds can be administered orally. The amount of a compound of thisinvention in the composition may vary widely depending on the type ofcomposition, size of a unit dosage, kind of excipients, and otherfactors well known to those of ordinary skill in the art. In general,the final composition may comprise from 5 mg to 50 mg of cPG for atypical adult weighing 50 to 120 kg or 1×10⁻⁵ percent to 3×10⁻⁴ byweight (% w) with the remainder being the excipient or excipients.

Other convenient administration routes include subcutaneous injection orintravenous infusion (for example the active cPG is dissolved in aphysiologically compatible carrier such as 0.9% sodium chloride ordextrose) or direct administration to the CNS. Using stereotacticdevices and accurate maps of an animals' CNS, a compound can be injecteddirectly into a site of neural damage.

The effective amount of compound in the CNS can be increased byadministration of a pro-drug form of a compound, which comprises acompound of the invention and a carrier, where the carrier is joined toa compound of the invention by a linkage which is susceptible tocleavage or digestion within the patient. Any suitable linkage can beemployed which will be cleaved or digested following administration.

However, there is no intention on the part of the applicants to excludeother forms of administration.

In other embodiments of the present invention, restoring nerve functionin an animal can include administering a therapeutic amount of cyclicProlyl Glycine or cPG compounds in combination with anotherneuroprotective agent, selected from, for example, growth factors andassociated derivatives (insulin-like growth factor-1 (IGF-I),insulin-like growth factor-11 (IGF-II), transforming growth factor-61,activin, growth hormone, nerve growth factor, growth hormone bindingprotein, IGF-binding proteins, keratinocyte growth factor,androgen-induced growth factor. Additional members of the FGF familyinclude, for example, fibroblast growth factor homologous factor-1(FHF-1), FHF-2, FHF-3 and FHF-4, karatinocyte growth factor 2, brainderived growth factor, neurotrophin 3, and neurotrophin 4. Other formsof neuroprotective therapeutic agents include clomethiazole, kynurenicacid, Semax, tacrolimus; glutamate agonist such as, NPS1506, GV1505260,MK-801, GV150526; AMP A ants such as2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX);anti-inflammatory agents directed against the addressin MAdCAM-1 and/orits integrin α4 receptors (α4β1 and α4β7), such as anti-MAdCAM-1 mAbMECA-367 (ATCC accession no. HB-9478).

Cyclic Prolyl Glycine compounds can be suitably administered by asustained-release system. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, for example, films, or microcapsules.

For parenteral administration, in one embodiment cyclic Prolyl Glcyineor cPG compounds is formulated generally by mixing each at the desireddegree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically, or parenterally,acceptable carrier, for example, one that is non-toxic to recipients atthe dosages and concentrations employed and is compatible with otheringredients of the formulation.

Generally, the formulations are prepared by contacting cyclic ProlylGlycine or cPG compounds uniformly and intimately with liquid carriersor finely divided solid carriers or both. Then, if necessary, theproduct is shaped into the desired formulation. Preferably the carrieris a parenteral carrier, ore preferably a solution that is isotonic withthe blood of the recipient. Examples of such carrier vehicles includewater, saline, Ringer's solution, a buffered solution, and dextrosesolution. Non-aqueous vehicles such as fixed oils and ethyl oleate arealso useful herein.

Cyclic Prolyl Glycine or cPG compounds are typically formulated in suchvehicles at a pH of from or about 4.5 to 8. It will be understood thatuse of certain of the foregoing excipients, carriers, or stabilizerswill result in the formation of salts of the compound. The finalpreparation may be a stable liquid or lyophilized solid.

Formulations of cyclic Prolyl Glycine or cPG compounds in pharmaceuticalcompositions can also include adjuvants. Typical adjuvants which may beincorporated into tablets, capsules, and the like are a binder such asacacia, corn starch, or gelatin; an excipient such as microcrystallinecellulose; a disintegrating agent like corn starch or alginic acid; alubricant such as magnesium stearate; a sweetening agent such as sucroseor lactose; a flavouring agent such as peppermint, wintergreen, orcherry. When dosage forms are tablets, cyclic Prolyl Glycine or a cPGcompounds and compositions can include binders and optionally, a smoothcoating. When the dosage form is a capsule, in addition to the abovematerials, it may also contain a liquid carrier such as a fatty oil.Other materials of various types may be used as coatings or as modifiersof the physical form of the dosage unit. A syrup or elixir may containthe active compound, a sweetener such as sucrose, preservatives likepropyl paraben, a colouring agent, and a flavouring agent such ascherry. Sterile compositions for injection can be formulated accordingto conventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as water ornaturally occurring vegetable oil like sesame, peanut, or cottonseed oilor a synthetic fatty vehicle like ethyl oleate or the like may bedesired.

For injection, intraventricular administration and other invasive routesof administration, cyclic Prolyl Glycine or cPG compounds are preferablysterile. Sterility may be accomplished by any method known in the art,for example filtration through sterile filtration membranes (forexample, 0.2-micron membranes), therapeutic compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper able to be pierced bya hypodermic injection needle.

Preferred Aspects of the Present Invention

A first aspect of the present invention includes a method ofregenerating neurons and glia cell loss as a result of an insult frominjury or disease, including the steps of: a) providing a subject inneed of said regenerating neurons and glia cell loss; and b)administering to the subject cyclic Prolyl Glycine (cPG) or itsanalogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline(cPMeG), collectively called a cPG compound, or a combination thereof,in an effective amount to regenerate new neurons and glia; wherein theneurons are regenerated and the glia cell loss is regenerated in saidsubject; further wherein the cPG compound serves as a neurogenesis agentin the central nervous system; and further wherein the neurons and gliacell loss as a result of an insult from injury or disease areregenerated.

Regenerating neurons generally refers to the regrowth or repair ofneurons, glia, axons, myelin, or synapses. Researchers are developingnew tools to effectively control the process of neural injury anddegeneration and to create a microenvironment that enhances the capacityfor innate repair and the efficacy of other regeneration strategies,including neural cell replacement and neurorehabilitation. Experiment 7and Experiment 8 describe the method of regenerating neurons and gliacell loss as a result of an insult from injury or disease

Another aspect of the present invention includes wherein theadministration is in the form of a pharmaceutical composition includinga pharmaceutically acceptable carrier.

A further aspect of the present invention includes wherein the effectiveamount of cPG compound is from about 1 jig to about 100 mg per kg ofbody weight.

An additional aspect of the present invention includes wherein theadministration is in combination with artificial cerebrospinal fluid.

Another aspect of the present invention includes wherein theadministration is intravenous.

A further aspect of the present invention includes wherein theadministration is combined with a neuroprotective agent, insulin-likegrowth factor-I (IGF-I) or insulin growth-like factor-II (IGF-II).

An additional aspect of the present invention wherein the administrationis combined with an anti-inflammatory agent, anti-integrin alpha 4subunit reagents.

A second aspect of the present invention includes a method of repairingdamaged neurons and glia cell loss as a result of an insult from injuryor disease, including the step of: a) providing a subject in need ofsaid regenerating neurons and glia cell loss; and b) administering tothe subject cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, in aneffective amount to regenerate new neurons and glia; wherein the neuronsare regenerated and the glia cell loss is regenerated in said subject;wherein said cPG serves as a neurorescue agent in the central nervoussystem and further wherein the damaged neurons and glia cell loss as aresult of an insult from injury or disease are repaired.

Repairing damaged neurons generally refers to reconstructive techniquesor processes to repair damaged neurons to prevent neuronal and glialloss. The present invention describes methods that can repair damagedneurons and glia following traumatic, anoxic, infectious, andimmunological adverse effects. The old doctrine that axons cannot bemade to regenerate, and dead neurons cannot be replaced, is no longertenable. In particular, Experiment 7 and Experiment 8 describe therepairing process through neuronal, glial, and pharmacologicalinterventions.

Another aspect of the present invention includes wherein cyclic ProlylGlycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) or cyclicGlycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, is from about 1 μg to about 100 mg per kg of bodyweight.

A further aspect of the present invention includes wherein theadministration is in the form of a pharmaceutical composition includingpharmaceutically acceptable carrier thereof.

An additional aspect of the present invention includes wherein theadministration is in combination with artificial cerebrospinal fluid.

Another aspect of the present invention includes wherein theadministration is combined with a neuroprotective agent, insulin-likegrowth factor-I (IGF-I) or insulin growth-like factor-II (IGF-II).

A further aspect of the present invention includes herein theadministration is combined with an anti-inflammatory agent.

A third aspect of the present invention includes a method for relievingor alleviating of cognitive impairment caused by a disease, injury, orcondition in a mammal in need thereof, comprising: a) providing a mammalin need of relieving or alleviating of cognitive impairment caused by adisease, injury, or condition; and b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to saidmammal; wherein the disease is selected from the group consisting ofAlzheimer's disease, Huntington's disease, Lewy Body disease, Dementia,and multi-infarct dementia, memory loss, attention deficit symptomsassociated with Alzheimer disease, neurodegeneration associated withAlzheimer disease, dementia of mixed vascular origin, dementia ofdegenerative origin, pre-senile dementia, senile dementia, dementiaassociated with Parkinson's disease, progressive supranuclear palsy orcortical basal degeneration; further wherein the injury is selected fromthe group consisting of neurotoxic injury, cerebral hypoxia/ischemia,traumatic brain injury, coronary artery bypass surgery, where saidcondition is normal aging, age-related memory loss, memory impairment,cholinergic hypofunction, vascular narrowing or blockage in the brain,neuroinflammation, mild cognitive impairment, cerebral atrophy,frontotemporal lobar degeneration, Pick's disease, HIV infection, Down'ssyndrome, and loss of synaptic plasticity; further wherein said mammalis for relieved or alleviated of cognitive impairment caused by adisease, injury, or condition, including Alzheimer Disease, memory loss,attention deficit symptoms associated with Alzheimer disease,neurodegeneration associated with Alzheimer disease, dementia of mixedvascular origin, dementia of degenerative origin, pre-senile dementia,senile dementia, dementia associated with Parkinson's disease,progressive supranuclear palsy or cortical basal degeneration.

Relieving cognitive impairment generally refers to methods that canrelieve symptoms related to memory, thinking, language and other thoughtprocesses. In addition, they may also help with mood, agitation andother behavioral issues.

Another aspect of the present invention includes wherein said cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Prolific or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, includes an aqueous solution and one or morepharmaceutically acceptable excipients, additives, carriers oradjuvants.

A further aspect of the present invention includes wherein said cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, further includes one or more excipients,carriers, additives, adjuvants or binders in a tablet or capsule.

An additional aspect of the present invention includes wherein thedisorder is a mild cognitive impairment, Alzheimer Disease, memory loss,attention deficit symptoms associated with Alzheimer disease,neurodegeneration associated with Alzheimer disease, dementia of mixedvascular origin, dementia of degenerative origin, pre-senile dementia,senile dementia, dementia associated with Parkinson's disease,progressive supranuclear palsy or cortical basal degeneration.

Another aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, is administered via an oral, intraperitoneal,intravascular, peripheral circulation, subcutaneous, intraorbital,ophthalmic, intraspinal, intracisternal, topical, infusion, implant,aerosol, inhalation, scarification, intracapsular, intramuscular,intranasal, buccal, transdermal, pulmonary, rectal, vaginal, or acombination thereof, route of administration.

Another aspect of the present invention includes wherein thepharmaceutically effective amount has a lower limit of about 0.001milligrams per kilogram mass (mg/kg) of said mammal and an upper limitof about 100 mg/kg of said mammal.

A further aspect of the present invention includes wherein the cognitiveimpairment is caused by cholinergic hypofunction.

An additional aspect of the present invention includes wherein thecognitive impairment is caused by a decrease in glutamate receptors inthe granular cell layer (CA1) of the hippocampus of said mammal.

Another aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, causes an increase in AMPA receptors in thegranular cell layer (CA1) of the hippocampus of said mammal.

A further aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, causes an increase in neuronal plasticity causedby said cPG compound in the granule cell layer (CA1) and the pyramidalcell layer (CAS) regions of said mammal's hippocampus.

An additional aspect of the present invention includes wherein thecerebral hypoxia/ischemia caused by traumatic brain injury.

Another aspect of the present invention includes wherein the cognitiveimpairment caused by multi-infarct dementia.

A further aspect of the present invention includes wherein the cognitiveimpairment caused by coronary arterial bypass surgery (CABG).

An additional aspect of the present invention includes wherein thecognitive impairment caused by Alzheimer Disease, memory loss, attentiondeficit symptoms associated with Alzheimer disease, neurodegenerationassociated with Alzheimer disease, dementia of mixed vascular origin,dementia of degenerative origin, pre-senile dementia, senile dementia,dementia associated with Parkinson's disease, progressive supranuclearpalsy or cortical basal degeneration.

A fourth aspect of the present invention includes a method of preventingthe symptoms of a mild cognitive impairment caused by or associated witha disease, injury, or condition in a mammal in need thereof, including:a) providing a mammal in need of preventing cognitive impairment causedby a disease, injury, or condition; b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to themammal; wherein the disease is selected from the group consisting ofAlzheimer's disease, Huntington's disease, Lewy Body disease, Dementia,and multi-infarct dementia, memory loss, attention deficit symptomsassociated with Alzheimers disease, neurodegeneration associated withAlzheimer disease, dementia of mixed vascular origin, dementia ofdegenerative origin, pre-senile dementia, senile dementia, dementiaassociated with Parkinson's disease, progressive supranuclear palsy orcortical basal degeneration.

Preventing the symptoms of a mild cognitive impairment generally refersactions that would be performed to prevent the symptomatic phase andprimarily to reduce the risk of disease. Typically, prevention indementia can be referred to as levels of prevention: primary, secondaryand tertiary.

In degenerative dementias, the secondary prevention stage can apply tothe phase of mild cognitive impairment. At this point, symptoms arepresent but are not sufficiently severe to constitute dementia.Therefore, treatment of cPG compounds aimed at subjects with MCI couldbe considered secondary prevention studies.

Tertiary prevention refers to a treatment designed to halt theprogression of the disease once it has been established. The goal is toreduce the disability and improve the long-term prognosis forindividuals with the mild cognitive impairment.

A fifth aspect of the present invention includes a method of treatingthe symptoms of a mild cognitive impairment caused by or associated witha disease, injury, or condition in a mammal in need thereof, including:a) providing a mammal in need of treating of cognitive impairment causedby a disease, Injury, or condition; b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or Its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Pro line or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to themammal; wherein the disease is selected from the group consisting ofAlzheimer's disease, Huntington's disease, Lewy Body disease, Dementia,and multi-infarct dementia, memory loss, attention deficit symptomsassociated with Alzheimer disease, neurodegeneration associated withAlzheimer disease, dementia of mixed vascular origin, dementia ofdegenerative origin, pre-senile dementia, senile dementia, dementiaassociated with Parkinson's disease, progressive supranuclear palsy orcortical basal degeneration.

Treating the symptoms of a mild cognitive impairment generally refers totreating cognitive function, or the process of thought, includes but isnot limited to abilities such as learning, reading, speaking andwriting. Patients with mild cognitive impairment (MCI) retain theseimportant cognitive skills, necessary to manage their everydayactivities, but have difficulty remembering recent events or recentlyacquired information. Long-term memories tend to remain intact. Inparticular, see Experiment 12, Clinical Trial on Alzheimer's Patientswith Mild Cognitive Impairment.

Neurons, are among the most ancient of all specialized animal celltypes. Their structure is like that of no other class of cells. Thecentral challenge of neural growth development is how to cause axons anddendrites grow out, find their right partners, and synapse with themselectively to create a functional network.

A sixth aspect of the present invention includes a method of increasingneuronal growth or synapse formation in a mammal caused by or associatedwith a disease, injury, or condition in a mammal in need thereofincluding: a) providing a mammal in need of increasing neuronal growthor synapse formation; b) administering an effective amount of acomposition that comprising a pharmaceutically effective amount ofcyclic Prolyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine)or cyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline orcyclic Glycyl-2-Methyl-Proline (cPMeG), collectively called a cPG, or acombination thereof, to the mammal; wherein the disease is selected fromthe group consisting of Alzheimer's disease, Huntington's disease, LewyBody disease, Dementia, and multi-infarct dementia, memory loss,attention deficit symptoms associated with Alzheimer disease,neurodegeneration associated with Alzheimer disease, dementia of mixedvascular origin, dementia of degenerative origin, pre-senile dementia,senile dementia, dementia associated with Parkinson's disease,progressive supranuclear palsy or cortical basal degeneration A seventhaspect of the present invention includes a method for relieving oralleviating of cognitive impairment caused by a disease, injury, orcondition in a mammal in need thereof, including: a) providing a mammalin need of relieving or alleviating of cognitive impairment caused by adisease, injury, or condition; and b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to themammal; wherein the disease is selected from the group consisting ofAlzheimer's disease, Huntington's disease, Lewy Body disease, Dementia,and multi-infarct dementia, memory loss, attention deficit symptomsassociated with Alzheimer disease, neurodegeneration associated withAlzheimer disease, dementia of mixed vascular origin, dementia ofdegenerative origin, pre-senile dementia, senile dementia, dementiaassociated with Parkinson's disease, progressive supranuclear palsy orcortical basal degeneration; further wherein the injury is selected fromthe group consisting of neurotoxic injury, cerebral hypoxia/ischemia,traumatic brain injury, coronary artery bypass surgery, where saidcondition is normal aging, age-related memory loss, memory impairment,cholinergic hypofunction, vascular narrowing or blockage in the brain,neuroinflammation, mild cognitive impairment, cerebral atrophy,frontotemporal lobar degeneration, Pick's disease, HIV infection, Down'ssyndrome, and loss of synaptic plasticity; further wherein said mammalis for relieved or alleviated of cognitive impairment caused by adisease, injury, or condition, including Alzheimer Disease, memory loss,attention deficit symptoms associated with Alzheimer disease,neurodegeneration associated with Alzheimer disease, dementia of mixedvascular origin, dementia of degenerative origin, pre-senile dementia,senile dementia, dementia associated with Parkinson's disease,progressive supranuclear palsy or cortical basal degeneration.

Another aspect of the present invention Includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, comprises an aqueous solution and one or morepharmaceutically acceptable excipients, additives, carriers oradjuvants.

A further aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, further comprises one or more excipients,carriers, additives, adjuvants or binders in a tablet or capsule.

An additional aspect of the present invention includes wherein thedisorder is a mild cognitive impairment, Alzheimer Disease, memory loss,attention deficit symptoms associated with Alzheimer disease,neurodegeneration associated with Alzheimer disease, dementia of mixedvascular origin, dementia of degenerative origin, pre-senile dementia,senile dementia, dementia associated with Parkinson's disease,progressive supranuclear palsy or cortical basal degeneration.

Another aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, is administered via an oral, intraperitoneal,intravascular, peripheral circulation, subcutaneous, intraorbital,ophthalmic, intraspinal, intracisternal, topical, infusion, implant,aerosol, inhalation, scarification, intracapsular, intramuscular,intranasal, buccal, transdermal, pulmonary, rectal, vaginal, or acombination thereof, route of administration.

A further aspect of the present invention includes wherein thepharmaceutically effective amount has a lower limit of about 0.001milligrams per kilogram mass (mg/kg) of said mammal and an upper limitof about 100 mg/kg of said mammal.

An additional aspect of the present invention includes wherein thecognitive impairment is caused by cholinergic hypofunction.

Another aspect of the present invention includes wherein the cholinergichypofunction is caused by scopolamine.

A further aspect of the present invention includes wherein the cognitiveimpairment is age-related memory loss, cognitive impairment, MCI (“mildcognitive impairment”), Alzheimer Disease, memory loss, attentiondeficit symptoms associated with Alzheimer disease, neurodegenerationassociated with Alzheimer disease, dementia of mixed vascular origin,dementia of degenerative origin, pre-senile dementia, senile dementia,dementia associated with Parkinson's disease, progressive supranuclearpalsy or cortical basal degeneration.

An additional aspect of the present invention includes wherein thecognitive impairment is caused by a decrease in glutamate receptors inthe granular cell layer (CA1) of the hippocampus of said mammal.

Another aspect of the present invention includes wherein the cPGcompound causes an increase in AMPA receptors in the granular cell layer(CA1) of the hippocampus of said mammal.

A further aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, causes an increase in neuronal plasticity causedby said cPG compound in the granule cell layer (CA1) and the pyramidalcell layer (CAS) regions of said mammal's hippocampus.

An additional aspect of the present invention includes wherein thecerebral hypoxia/ischemia caused by traumatic brain injury.

Another aspect of the present invention includes wherein the cognitiveimpairment caused by multi-infarct dementia.

A further aspect of the present invention includes wherein the cognitiveimpairment caused by coronary arterial bypass surgery (CABG).

An additional aspect of the present invention includes wherein thecognitive impairment caused by Alzheimer Disease, memory loss, attentiondeficit symptoms associated with Alzheimer disease, neurodegenerationassociated with Alzheimer disease, dementia of mixed vascular origin,dementia of degenerative origin, pre-senile dementia, senile dementia,dementia associated with Parkinson's disease, progressive supranuclearpalsy or cortical basal degeneration.

An eight aspect of the present invention includes a method of preventingthe symptoms of a mild cognitive impairment caused by or associated witha disease, injury, or condition in a mammal in need thereof, including:a) providing a mammal in need of preventing cognitive impairment causedby a disease, injury, or condition; and b) administering apharmaceutically effective amount of cyclic Prolyl Glycine (cPG) or itsanalogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline(cPMeG), collectively called a cPG, or a combination thereof, to themammal; wherein the disease is selected from the group consisting ofAlzheimer's disease, Huntington's disease, Lewy Body disease, Dementia,and multi-infarct dementia, memory loss, attention deficit symptomsassociated with Alzheimer disease, neurodegeneration associated withAlzheimer disease, dementia of mixed vascular origin, dementia ofdegenerative origin, pre-senile dementia, senile dementia, dementiaassociated with Parkinson's disease, progressive supranuclear palsy orcortical basal degeneration.

A ninth aspect of the present invention includes a method of treatingthe symptoms of a mild cognitive impairment caused by or associated witha disease, injury, or condition in a mammal in need thereof, including:a) providing a mammal in need of treating of cognitive impairment causedby a disease, injury, or condition; and b) administering apharmaceutically effective amount of cyclic Prolyl Glycine (cPG) or itsanalogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline(cPMeG), collectively called a cPG compound, or a combination thereof,to the mammal; wherein the disease is selected from the group consistingof Alzheimer's disease, Huntington's disease, Lewy Body disease,Dementia, and multi-infarct dementia, memory loss, attention deficitsymptoms associated with Alzheimer disease, neurodegeneration associatedwith Alzheimer disease, dementia of mixed vascular origin, dementia ofdegenerative origin, pre-senile dementia, senile dementia, dementiaassociated with Parkinson's disease, progressive supranuclear palsy orcortical basal degeneration.

A tenth aspect of the present invention includes a method of increasingneuronal growth or synapse formation in a mammal caused by or associatedwith a disease, injury, or condition in a mammal in need thereofincluding: a) providing a mammal in need of increasing neuronal growthor synapse formation; and b) administering an effective amount of acomposition that comprising a pharmaceutically effective amount ofcyclic Prolyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine)or cyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline orcyclic Glycyl-2-Methyl-Proline (cPMeG), collectively called a cPGcompound, or a combination thereof, to the mammal; wherein the diseaseis selected from the group consisting of Alzheimer's disease,Huntington's disease, Lewy Body disease, Dementia, and multi-infarctdementia, memory loss, attention deficit symptoms associated withAlzheimer disease, neurodegeneration associated with Alzheimer disease,dementia of mixed vascular origin, dementia of degenerative origin,pre-senile dementia, senile dementia, dementia associated withParkinson's disease, progressive supranuclear palsy or cortical basaldegeneration.

An eleventh aspect of the present invention includes a method formodulating neurogenesis in neural tissue of a patient exhibiting atleast one symptom of a central nervous system disorder that isneurodegenerative disorders, ischemic disorders, neurological traumas,learning and memory disorders, or a combination thereof, byadministrating cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, or itsfunctional or active analogs, variants, derivatives, that have the same,substantially the same, or similar function as cPG, and combinationsthereof, wherein the agent modulates neurogenesis in the patient,thereby modulating neurogenesis in the neural tissue of the patient.

Neurogenesis is the process by which neve neurons arise from neural stemand progenitor cells, mature, specialize and become integrated andfunctional within the neuronal network. Modulating neurogenesis inneural tissue generally refers to method to exert a modifying orcontrolling influence on the neuronal growth in the human brain.

Another aspect or the present invention includes wherein the nervoussystem disorder is Alzheimer's disease, Parkinson's disease andParkinsonian disorders, Huntington's disease multiple sclerosis,amyotrophic lateral sclerosis, Shy-Drager syndrome, progressivesupranuclear palsy, Lewy body disease, spinal ischemia, ischemic stroke,cerebral infarction, spinal cord injury, and cancer-related brain andspinal cord injury, multi-infarct dementia, geriatric dementia, mildcognition impairment, depression and traumatic injury.

A further aspect of the present invention includes wherein themodulating neurogenesis is performed by an activation of a GPCR receptorin said neural tissue.

An additional aspect of the present invention includes wherein the agentis administered at an amount of cyclic Prolyl Glycine (cPG) or itsanalogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline(cPMeG), collectively called a cPG compound, or a combination thereof,from about 0.1 mg to about 10 mg/kg per day, from about 0.5 mg to about20 mg/kg per day, from about 0.2 mg to about 40 mg/kg per day, fromabout 5 mg to about 50 mg/kg per day, or from about 10 micrograms toabout 100 mg/kg per day.

An twelfth aspect of the present invention includes a method formodulating neurogenesis in neural tissue of a patient exhibiting atleast one symptom of a central nervous system disorder that isneurodegenerative disorders, ischemic disorders, neurological traumas,learning and memory disorders, or a combination thereof, byadministrating said patient an amount of cyclic Prolyl Glycine (cPG) orits analogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-AllylProline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, sufficient to increase adult neural stem cells insaid patient and reduce at least one symptom of said disorder.

Another aspect of the present invention includes wherein the disorderis, Alzheimer's disease, Parkinson's disease and Parkinsonian disorders,Huntington's disease multiple sclerosis, amyotrophic lateral sclerosis,Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease,spinal ischemia, ischemic stroke, cerebral infarction, spinal cordinjury, and cancer-related brain and spinal cord injury, multi-infarctdementia, geriatric dementia, cognition impairment, depression,traumatic injury, or a combination thereof.

Another aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, is administered from about 0.01 mg/kg to about100 mg per kilogram of body weight per day.

A thirteenth aspect of the present invention includes a method fortreating depression or other psychological disorder in a subject,including; a) providing a subject in need of treatment of depression orother psychological disorders; and b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to thesubject; wherein the subject is treated for depression or otherpsychological disorders.

Depression symptoms can vary from mild to severe and can include:feeling sad or having a depressed mood, loss of interest or pleasure inactivities once enjoyed, changes in appetite—weight loss or gainunrelated to dieting, trouble sleeping or sleeping too much, loss ofenergy or increased fatigue, difficulty thinking, concentrating ormaking decisions, and thoughts of death or suicide.

Another aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, comprises an aqueous solution and one or morepharmaceutically acceptable excipients, additives, carriers oradjuvants.

A further aspect of the present invention includes wherein the cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof further comprises one or more excipients, earners,additives, adjuvants or binders in a tablet or capsule.

An additional aspect of the present invention includes wherein thecyclic Prolyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine)or cyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline orcyclic Glycyl-2-Methyl-Proline (cPMeG), collectively called a cPGcompound, or a combination thereof, is administered via an oral,intraperitoneal, intravascular, peripheral circulation, subcutaneous,intraorbital, ophthalmic, intraspinal, intracisternal, topical,infusion, implant, aerosol, inhalation, scarification, intracapsular,intramuscular, intranasal, buccal, transdermal, pulmonary, rectal,vaginal, or a combination thereof, route of administration.

Another aspect of the present invention includes wherein thepharmaceutically effective amount has a lower limit of about 0.1milligrams per kilogram mass (mg/kg) of said mammal and an upper limitof about 10 mg/kg of said mammal.

A further aspect of the present invention includes wherein thepharmaceutically effective amount is between about 20 mg and about 80 mgper day given orally and can be up to 100 mg per day for some severecases per physician's prescription order.

A thirteenth aspect of the present invention includes a method ofpreventing the symptoms of depression or other psychological disordersin subject in need thereof, including: a) providing a subject in need ofpreventing depression or other psychological disorders; b) administeringa pharmaceutically effective amount of cyclic Prolyl Glycine (cPG) orits analogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-AllylProline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, to the subject; wherein the subject is preventedfrom developing the symptoms of depression or other psychologicaldisorders.

Preventing the symptoms of depression generally refers to the preventionof medical illness that negatively affects how the person feels, thinksand acts. Depression causes feelings of sadness and/or a loss ofinterest in activities once enjoyed. It can lead to a variety ofemotional and physical problems and can decrease a person's ability tofunction at work and at home. In particular, see Experiment 15.

A fourteenth aspect of the present invention includes a method oftreating the symptoms of depression or other psychological disorders ina subject in need thereof, including: a) providing a subject in need oftreating of depression or other psychological disorders; b)administering a pharmaceutically effective amount of cyclic ProlylGlycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) or cyclicGlycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), collectively called a cPG compound, ora combination thereof, to the mammal; wherein the subject is treated forthe symptoms of depression or other psychological disorders.

Depression symptoms can vary from mild to severe and can include:feeling sad or having a depressed mood, loss of interest or pleasure inactivities once enjoyed, changes in appetite—weight loss or gainunrelated to dieting, trouble sleeping or sleeping too much, loss ofenergy or increased fatigue, difficulty thinking, concentrating ormaking decisions, and thoughts of death or suicide.

A fifteenth aspect of the present invention includes a method for thetreatment of mentally depressed patients, including: a) providing asubject in need of treating of depression or other psychologicaldisorders; b) administering to a patient suffering from mentaldepression a pharmaceutically effective amount (such as but not limitedto from 20 mg to 80 mg per day) of cyclic Prolyl Glycine (cPG) or itsanalogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline(cPMeG), collectively called a cPG compound, or a combination thereof,wherein the subject is treated for mental depression.

Treating of mentally depressed patients has generally referred to usingantidepressants available in the market which normally increase the riskof suicidal thoughts and behavior in children, adolescents, and youngadults in short-term studies. In the present invention, cPG has beendemonstrated to serve as an effective an antidepressant with little orno adverse effects, see Experiment 15.

A sixteenth aspect of the present invention includes a method forrelieving or alleviating the the symptoms of depression or otherpsychological disorders in a subject in need thereof, including: a)providing a subject in need of relieving or alleviating depression orother psychological disorders; b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to themammal; wherein the subject is alleviated for the symptoms of depressionor other psychological disorders.

A seventeenth aspect of the present Invention includes a method forintervening or prevention the cascade of depression or otherpsychological disorders in a subject in need thereof, including: a)providing a subject in need of intervention or prevention of depressionor other psychological disorders; b) administering a pharmaceuticallyeffective amount of cyclic Prolyl Glycine (cPG) or Its analogues(cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline, or cyclicGlycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline (cPMeG),collectively called a cPG compound, or a combination thereof, to themammal; wherein said the subject is prevented for the symptoms ofdepression or other psychological disorders.

While well-established treatments exist for depression, estimatessuggest a majority of persons with depression do not receive appropriatecare. The timely intervention is an Important element of the continuumof care model to person with Major Depressive Episodes. In particular,see Experiment 15.

EXAMPLES

The following in vitro and in-vivo studies demonstrate efficacy ofcyclic Prolyl Glycine in reducing cognitive impairment. They are notintended to be limiting, and other compositions and methods of thisinvention can be developed without undue experimentation. All thefollowing experiments were carried out using protocols developed underguidelines approved by the Animal Ethics Committee and InstitutionalReview Board.

Cyclic Prolyl Glycine is available from commercial suppliers such asBachem (Torrance, Calif.), and Sigma (St. Louis, Mo.),

Experiment 1

Cyclic PG Prevents Glutamate Induced Neuronal Death In Vitro in a DoseRelated Manner.

Materials and Methods: Cerebellar Cell Culture Preparing and Coating ofCover Slips

Ten coverslips were placed into a large petri dish and washed in 70%alcohol for 5 minutes, then washed with Millipore H₂O. The coverslipswere air dried, then coated with Poly-D-Lysine (1 mg/ml stock solutionin PBS, 90-100 μl) and incubated for 2 hours at 34° C.

Extraction

Postnatal day 4 Wistar rats were used for the study. Rats were placed inice for 1 minute, the heads were decapitated and the cerebellum removedon ice. Cerebellum tissue was placed in 1 ml of 0.65% glucosesupplemented PBS (10 μl 65% stock D (+)glucose/l ml PBS) in a largepetri dish, chopped up into smaller sections and triturate with a 1 mlinsulin syringe via a 23 G (0.4 mm) needle, and then squirted back intothe glucose solution on the large petri dish. The tissue was sievedthrough (125 μm pore size gaze) and centrifuged (2 minutes at 60 g) twotimes for a medium exchange into serum-free BSA-supplemented START Vmedium (Biochrom). The second centrifugation step was done with 1 ml ofSTART V medium. The microexplants were reconstituted into 500 μl ofSTART V medium and put on ice.

Cultivation and Fixation of Cerebellar Cells

Two hours after PDL-coating, the slides were washed with Millipore H₂Oand air dried. Each slide was placed into a small 35 mrn petri dish and40 μl of START V/cell suspension added. The tissue was incubated for 2hours at 34° C. (settlement period). START V-medium (1 ml) was thenadded to the petri dish and cultivated at 34° C./5% CO2/100% humidityfor 48 hours. Cells were rinsed in PBS and then fixed for 2-3 minutes inincreasing concentrations of paraformaldehyde (500 μl of 0.4% PFA wasapplied; then 1.2% PFA; then 3% PFA and finally 4% PFA—all fixationsolutions contain 0.2% glutardialdehyde). Finally, the microexplantswere rinsed in PBS.

Drug Application

10 μl of toxin (L-glutamate-100 mM in Millipore water) was appliedsimultaneously with cPG (from bachem, 10 mM stock prepared in PBS anddiluted to final concentrations between 1-100 nM) for Study 1. A delayin administration of cPG at 6 hours after glutamate treatment wasperformed for Study 2.

Result:

Study 1: Glutamate treatment resulted in 85% loss of cerebellum neurons.Cyclic PG significantly reduced the glutamate induced neuronal death ina dose response manner when administered simultaneously with glutamate(FIG. 3). The treatments with lower doses of cPG (10-100 nM) showedsignificant recovery from glutamate-induced neurotoxicity.

Study 2: Cyclic PG showed a significantly recovery from glutamateinduced neurotoxicity in a dose range of 1-100 nM when given 6 hoursafter the glutamate treatment compared to the vehicle treated group(FIG. 4).

A further lower dose of cPG also showed a significant increase in neuronnumber compared to the normal control group, suggest a role for cPG inneuronal proliferation and differentiation.

Conclusions

Excessive glutamate can cause neuronal excitotoxicity by active NMDAreceptors. cPG completely prevented the glutamate-induced neurotoxicity,when given either immediately or 6 hours after the glutamate treatmentby acting as a direct or indirect NMDA agonist. Given that cPG canagonise mGlu2/3 receptor, which can inhibit NMDA activity. GPE, thepre-hormone for cPG has been shown to be partial NMDA receptor inpromoting pCREB, probably due to its antic effect on mGlu2/3 receptors.cPG can be involved in preventing neurons undergoing apoptosis becausecPG can be effective as a delayed treatment, and promoted the neuronalproliferation.

Experiment 2

Cyclic(tri(prolylglycyl)) or c(PG)3 Prevents Glutamate Induced NeuronalDeath In Vitro in a Dose Related Manner.

Materials and Methods: (see above for Experiment 1, which isincorporated by reference herein.)

Drug Application

10 μl of toxin (L-glutamate-100 mM in Millipore water) was appliedsimultaneously with cyclic(tri(prolylglycyl)) (which was obtained fromNeuroBiomed, San Jose, Calif.), 10 mM stock prepared in PBS and dilutedto final concentrations between 1-100 nM) for Study 1. A delay inadministration of cyclic(tri(prolylglycyl)) at 6 hours after glutamatetreatment was performed for Study 2.

Result:

Study 1: Glutamate treatment resulted in 85% loss of cerebellum neurons.cyclic(tri(prolylglycyl)) significantly reduced the glutamate inducedneuronal death by 57% in a dose response manner when administeredsimultaneously with glutamate. The treatments with lower doses ofcyclic(tri(prolylglycyl)) (10-100 nM) showed significant recovery fromglutamate-induced neurotoxicity.

Study 2: cyclic(tri(prolylglycyl)) showed an improvement ofapproximately 43% significantly recovery from glutamate inducedneurotoxicity in a dose range of 1-100 nM when given 6 hours after theglutamate treatment compared to the vehicle treated group.

A further lower dose of cyclic(tri(prolylglycyl)) also showed asignificant increase in neuron number compared to the normal controlgroup, suggest a role for cPG in neuronal proliferation anddifferentiation.

Experiment 3

Cyclic Glycyl-2-Methyl Proline Prevents Glutamate Induced Neuronal DeathIn Vitro in a Dose Related Manner.

Materials and Methods: (see above for Experiment 1 and Experiment 2,which is incorporated by reference herein)

Drug Application

10 μl of toxin (L-glutamate-100 mM in Millipore water) was appliedsimultaneously with cyclic Glycyl-2-Methyl Proline (obtainedNeuroBiomed, San Jose Calif.), 10 mM stock prepared in PBS and dilutedto final concentrations between 1-100 nM) for Study 1. A delay inadministration of cPG at 6 hours after glutamate treatment was performedfor Study 2.

Result:

Study 1: Glutamate treatment resulted in 85% loss of cerebellum neurons,cyclic Glycyl-2-Methyl Proline significantly reduced the glutamateinduced neuronal death by 63% in a dose response manner whenadministered simultaneously with glutamate. The treatments with lowerdoses of cyclic Glycyl-2-Methyl Pro line (10-100 nM) showed significantrecovery from glutamate-induced neurotoxicity.

Study 2: cyclic Glycyl-2-Methyl Proline showed an improvement ofapproximately 58% significantly recovery from glutamate inducedneurotoxicity in a dose range of 1-100 nM when given 6 hours after theglutamate treatment compared to the vehicle treated group.

A further lower dose of cyclic Glycyl-2-Methyl Proline also showed asignificant increase in neuron number compared to the normal controlgroup, suggest a role for cPG in neuronal proliferation anddifferentiation.

Conclusions

Excessive glutamate can cause neuronal excitotoxicity by active NMDAreceptors. Cyclic PG analogues, cyclic(tri(prolylglycyl)) and cyclicGlycyl-2-Methyl Proline significantly prevented the glutamate-inducedneurotoxicity, when given either immediately or 6 hours after theglutamate treatment by acting as a direct or indirect NMDA agonist.

Cyclic PG and its analogues, such as but not limited tocyclic(tri(prolylglycyl)) and cyclic Glycyl-2-Methyl Proline can be usedin preventing neurons undergoing apoptosis because cPG compounds can beeffective as a delayed treatment, and promoted the neuronalproliferation.

Experiment 4

Effects of cPG after 6-OHDA Induced Nigral-Striatal Lesion.

Materials and Methods

Twenty male Wistar rats (280-310 g) were used. After exposing the skull,6-OHDA (8 jag in abase of 2 μl 0.9% saline containing 1% ascorbic acid)was administered into the right medial forebrain bundle (MFB) usingco-ordinates AP+4.7 mm, R 1.6 mmv-8 mm under 3% halothane anaesthesia.6-OHDA was injected through a 25G needle connected via a polyethylenecatheter to a 100 μl Hamilton syringe. The 6-OHBA was infused by amicrodialysis infusion pump at a rate of 0.5 μl/min. The needle was leftin the brain for a further 3 minutes before being slowly withdrawn. Theskin was sutured with 2.0 silk and the rats were allowed to recover fromanaesthesia. The rats were housed in a holding room with free access tofood and water at all times except during behavioural testing.

Cyclic PG was dissolved in saline. Four different doses of cPG (0, 0.1,0.5, 1 mg/kg, Bachem) were administered intraperitoneally 2 hours postlesion.

At 7 days post-lesion, rats were injected with 0.1 mg/kg apomorphine andthe number of contralateral rotations/hour was recorded and calculatedusing a computerised Rotameter (St Diego Instruments). Experimenter wasblinded from the treatment groups.

Result:

The group treated with 1 mg cPG (n=5, 154±64) showed a trend toward areduction in the number of rotations compared to the vehicle treatedgroup (n=5, 290±18) indicating that cPG in improves functional recoveryin 6-OHDA induced nigrostriatal injury. (FIG. 5).

Experiment 5

Effects of Cyclic(tri(prolylglycyl)) After 6-OHDA InducedNigral-Striatal Lesion.

Materials and Methods (see above for Experiment 1, Experiment 2,Experiment 3, and Experiment 4, which is incorporated by referenceherein)

Cyclic(tri(prolylglycyl)) was dissolved in saline solution. Fourdifferent doses of Cyclic(tri(prolylglycyl)) (0, 0.1, 0.5, 1 mg/kg,NeuroBiomed) were administered intraperitoneally 2 hours post lesion.

The group treated with 1 mg cyclic(tri(prolylglycyl)) (n=5, 172±69)showed a trend toward a reduction in the number of rotations compared tothe vehicle treated group (n=5, 290±18) indicating a role forcyclic(tri(prolylglycyl)) in improving functional recovery in 6-OHDAinduced nigrostriatal injury.

Conclusions

Cyclic(tri(prolylglycyl)) improved the functional recovery after 6-OHDAinduced nigral-striatal lesions in a dose related manner.

This data indicates Cyclic(tri(prolylglycyl)) can be used as a treatmentfor Parkinson's disease and other neurological disorders.

Experiment 6

Effects of Cyclic Glycyl-2-Methyl Proline After 6-OHDA InducedNigral-Striatal Lesion.

Materials and Methods (see above for Experiment 1 through Experiment 5,which are incorporated by reference herein).

Cyclic Glycyl-2-Methyl Proline (cGMeP) was dissolved in saline solution.Four different doses of cGMeP (0, 0.1, 0.5, 1 mg/kg, NeuroBiomed) wereadministered intraperitoneally 2 hours post lesion.

The group treated with 1 mg cGMeP (n=5, 134±69) showed a trend toward asignificant reduction in the number of rotations compared to the vehicletreated group (n=5, 292±21) indicating a role for cGMeP in improvingfunctional recovery in 6-OHDA induced nigrostriatal injury.

Conclusions

Cyclic Glycyl-2-Methyl Proline improved the functional recovery after6-OHDA induced nigral-striatal lesions in a dose related manner.

This data indicated that Cyclic Glycyl-2-Methyl Proline has efficacy asa treatment for Parkinson's disease.

Experiment 7

Morris Water Maze (MWM) Model of Learning and Memory Used to AssessEffects of Cyclic Prolyl Glycine on Cognitive Function.

cPG administered to animals treated with scopolamine-induced cognitivedysfunction produces clinical improvement in those animals, similar tothe therapeutic improvement observed in people suffering fromcholinergic hypofunction. Scopolamine is commonly used in animal modelsof cholinergic hypofunction associated with Alzheimer's disease. Thefunctional deficits observed after scopolamine treatment include thosefound in human patients with Alzheimer's disease. Thus, scopolaminetreatment is reasonably predictive of cognitive Impairment found inhuman diseases. Additionally, scopolamine treatment mimics cognitivedisfunctions in humans who do not have neurodegenerative disorders.

The purpose of the study was to investigate cyclic Prolyl Glycine toevaluate its impact cognitive deficit and affective state (anxiety).

Methods

The first part of the study involved acute testing of the cyclic ProlylGlycine in the Morris Water Maze memory model. The MWM test is one ofthe most frequently used tests for assessing spatial memory in rats andis well recognized to accurately predict effects of disease andtreatment on spatial memory generally. Therefore, the MWM test reflectseffects of disease and treatment in human subjects.

The standard procedure for MWM was followed. We used a circular swimmingpool (HO cm depth×150 cm diameter) filled with opaque water, with thetemperature maintained at 20° C. A platform was hidden 1 cm below thewater surface, with a white flag (10 cm×10 cm) located either 20 cmabove the platform for the visual cue and at 3 o'clock position inrelation to the starting location for a spatial cue. On days 1-4 of theexperiment rats underwent memory acquisition trials with 6 trials (60seconds each) in each day of testing (habituation phase). Latency toreach the platform was recorded and the daily reduction of averagelatency was used to measure the capability to learn where the hiddenplatform was.

On day 5 of the experiment normal, non-aged Wistar rats were split intogroups to receive either saline (N=12) or scopolamine (0.5 mg/kg, i.p.,N=12) to induce memory deficit. Scopolamine was administered half anhour before the probe test commenced.

10 min following the scopolamine treatment, the cyclic Prolyl Glycinewas administered orally at 10 mg/kg (14=16) with vehicle-treated animalsadministered the diluent by oral gavage using an identical treatmentprotocol (n=15),

TABLE 1 Animals Used to Test Effects of cPG on Memory ScopolamineVehicle Vehicle N = 12 N = 12 cPG N = 16 N = 15

Acute effects of cPG were then tested in animals withscopolamine-induced memory impairment and in age-matched control animalswith no memory impairment to determine any direct pharmacological effecton memory processing. Experimental groups are detailed in the Table 1below.

On day 5, the probe MWM test was performed with the platform removed.There were 6 trials, each of maximum duration of 60 s, at least 5 minrest between trials). The amount of time the rats spend swimming nearthe platform provided a measure of how much they relied on visual andspatial cue to locate the platform, as opposed to using a non-spatialstrategy. Data was collected and analyzed using Any-maze (v4.2)software.

The data generated from behavioral tests was analyzed using one-wayANOVA for determining the difference between the aged-groups. Two-wayANOVA was used for examining the progress of behavioral results with thetime points treated as dependent factors. GraphPad Prism version 3.02was used for data analysis.

Results

Treatment with scopolamine significantly impaired acquisition of spatialmemory in treated animals (time to platform approximately 208% ofcontrol on day 4). Cyclic Prolyl Glycine (20 mg/kg; daily) significantlyreversed the cognitive impairment induced by scopolamine. (FIG. 6).

These results confirmed the presence of choline-positive effect inNA-831 on retrieval of learned skill of finding a submerged platform(spatial memory) and that this drug can be effective in patients withmild cognitive impairment.

Experiment 8

Determination of Neurogenesis by Testing cPG and its Analogues c(PG)3Anti cGMeP with Bromodeoxyuridine

Objective

The objective of this experiment is to study the impact ofintra-peritoneal infusion of cPG and its analogues c(PG)3 and cGMeP inrats, co-administrated with BrdU in highly neurogenic regions includingthe sub ventricular zone and the dentate gyrus in the hippocampus.

Experimental Method:

Male Wistar rats weighing about 250=270 g (not newborn) were used. Allanimal experiments were conducted in agreement with national andinternational guidelines. Care was taken to minimize suffering for theanimals. Animals were allowed to acclimatize for 1 week before start ofthe studies. Animals were housed under standardized conditions withnormal light-dark periods and in groups of five animals per cage.Animals had access to food and water ad libitum during the studies.

Three neurogenesis modulating agents, cPG, c(PG)3 and cGMeP wereseparately administered intraperitoneally to Male Wistar rats (N=10) at10 mg/kg in 0.1% Rat Serum Albumin (RSA). The negative control (n=12),the vehicle group was injected with saline (in 0.1% RSA).Bromodeoxyuridine (BrdU; 50 mg/kg) was co-administrated together withthe compounds. The intraperitoneally injections were given with a 12hour interval for 7 days. Animals were perfused on day 8. The rats werekept at 12 hours light/dark regime. In perfusion, animals were perfusedtranscranially with 50 ml of ice cold phosphate buffered saline (PBS)and then 100 ml of 4% paraformaldehyde in PBS. Brains were fixed afterremoval in 4% paraformaldehyde in PBS for 24 hours at 4° C., at least 3days before sectioning. The procedures of transcranial infusion consistthe following steps:

The animals were weighed to the nearest 0.1 grams and were administeredwith sodium pentobarbital and ketamine/xylazine. The animals were placedin a heated cage for 10-15 minutes. The rats were secured in the supineposition (lying on the back with face upward) with its forepaws and hindpaws pinned to a Styrofoam work surface inside a chemical fume hood. Anincision was made through the skin with surgical scissors along thethoracic midline from just beneath the xiphoid process to the clavicle.Two additional skin incisions were made from the xiphoid process alongthe base of the ventral rib cage laterally. Gently reflect the two flapsof skin rostrally and laterally making sure to expose the thoracic fieldcompletely. The cartilage of the xiphoid process was grasped with bluntforceps and it was raised slightly to insert pointed scissors. Thethoracic musculature and ribcage were cut through between the breastboneand medial rib insertion points and the incision was extended rostrallyto the level of the clavicles. The diaphragm from the chest wall on bothsides was separated with scissor cuts. The reflected ribcage was tapedor pinned with 18G needles laterally to expose the heart and otherthoracic organs. The the pericardial sac was gently grasped with bluntforceps and was tom open fully. The beating heart was secured with bluntforceps and a 1-2 mm incision was made in the left ventricle. A 24G×25.4mm animal feeding needle with a bulnous tip (Harvard apparatus Cat.#52-4009) was inserted. The feeding needle was thread into the base ofthe aortic arch using a dissecting microscope. The needle base wasclamped to the left ventricle above the incision site using a hemostat.The right atrium was cut immediately with scissors and at the first signof blood flow, the infusion of heparinized saline was started (stage 1perfusate) and continued until the fluid exiting the right atrium isentirely clear. The saline perfusate was changed to aldehyde-basedfixative (stage 2 perfusate) to a total of 20-30 ml of fixative asinfused to an animal. The animal was decapitated with large surgicalscissors. The brains were removed and embedded in paraffin. Sectionswere prepared using a freezing microtome and stored in cryoprotectant at−20° C. before immunostaining for BrdU. Sections were immunostained forBrdU with mouse anti-BrdU paired with a biotinylated goat anti mouse IgGand visualized using ABC Elite kit (Vectorlabs, using manufacturesdirections).

Standard light microscope techniques were used to count the total numberof BrdU positive cells in each section and in relevant region of thebrain. Analysis and quantification were performed for proliferativebrain regions, subventricular zone, and the dentate gyros inhippocampus. Other experimental details not listed here are known to oneof skill in the art and may be found for example in Pencea V et al. J.Neurosci Sep. 1 (2001). 21(17)16706-17.

Results

Notably, it was found that rats given intra-peritonea 1 infusion of cPG,c(PG)3 and cGMeP at 10 mg/kg in 0.1% RSA, co-administrated with BrdUtwice daily showed a significant increase (nonparametric One-way ANOVA)in the number of newborn cells (BrdU positive compared to sham injected)in highly neurogenic regions including the sub ventricular zone and thedentate gyrus in the hippocampus (FIG. 7 and FIG. 8).

Conclusion

In summary, the experiments demonstrated that cPG, c(PG)3 and cGMePexhibit neural stem cell proliferative effect pointing to neugenesis.

Experiment 9

Determination of the Regeneration of Damaged Nerve Tissue with ePG

Postnatal day 4 Wistar 10 rats were used for the study. The rats weredivided in 2 groups: one group of 5 animals treated with drug solution,and the other group of 5 animals treated with 0.9% sodium chloride(saline) solution. A drug solution containing cPG (from Bachem, 10 mMsolution prepared in 0.9% sodium chloride (saline) solution) wasadministered four times a day in volumes of 0.75 ml to thoroughly floodthe site of injury.

Two days after crashing the spinal cord, the dura of a rat was openedand a polyethylene tube was sutured to the vertebral spines and adjacentsoft tissues so that the opening in one end lay directly over theinjured part of the spinal cord. The tubing was brought through asubcutaneous tunnel so that its other end emerged at the base of theskull. A syringe adapter was attached to the external opening forinjecting the drugs. All experiments were on a double-blind basis on 2groups: one group of 5 animals treated with drug solution of 10 mM cPGin saline solution, and the other group of 5 animals treated with 0.9%sodium chloride (saline) solution. Treatment of every animal wascontinued for 14 days, after which the animals were killed andhistological sections prepared.

Results

The drug treated animals showed greater invasion of the lesion by nervefibers than did the vehicle treated with saline solution. In thedrug-treated animals, the nerve fibers grew into the lesion site in suchprofusion that they were no longer oriented longitudinally, but grewrather haphazardly in all directions. Fibers were frequently undulatingand varicose and were often arranged in small bundles containing 3-6axons. The axons were very fine in calibers, most of them being 3-7microns in diameter. When the slides were coded and randomized, therewas no difficulty in distinguishing between the specimens from thedrug-treated and the saline treated animals. The most prolific nervegrowth occurred in the animals treated with cPG.

Conclusion

This Example showed that nerve regeneration is promoted by thoroughlybathing or otherwise contacting the injury site with the foregoingcomposition. The foregoing composition promotes regeneration of damagednerve tissue when administered directly to the site of the injury.

Experiment 10

Regeneration of Damaged Nerve Tissue of Cyclic(Glycyl-L-prolylglycyl-L-prolylglycyl-L-prolyl)

Postnatal day 4 Wistar 10 rats were used for the study. The rats weredivided in 2 groups: one group of 5 animals treated with drug solution,and the other group of 5 animals treated with 0.9% sodium chloride(saline) solution. A drug solution containing 10 mM of cyclic(glycyl-L-prolylglycyl-L-prolylglycyl-L-prolyl) or c(PG)3, obtained fromNeuroBiomed, San Jose, Calif. 10 mM solution prepared in 0.9% sodiumchloride (saline) solution was administered four times a day in volumesof 0.75 ml to thoroughly flood the site of injury.

Two days after crushing the spinal cord, the dura of a rat was openedand a polyethylene tube was sutured to the vertebral spines and adjacentsoft tissues so that the opening in one end lay directly over theinjured part of the spinal cord. The tubing was brought through asubcutaneous tunnel so that its other end emerged at the base of theskull. A syringe adapter was attached to the external opening forinjecting the drugs. All experiments were on a double-blind basis on 2groups: one group of 5 animals treated with drug solution of 10 mMc(PG)3 in saline solution, and the other group of 5 animals treated with0.9% sodium chloride (saline) solution. Treatment of every animal wascontinued for 14 days, after which the animals were killed andhistological sections prepared.

Results

The results are similar to the above experiment with cPG. In the c(PG)treated animals, the nerve fibers grew into the lesion site in suchprofusion that they were no longer oriented longitudinally, but grewrather haphazardly in all directions. Fibers were frequently undulatingand varicose and were often arranged in small bundles containing 3-6axons. The axons were very fine in caliber, most of them being 3-7microns in diameter.

When the slides were coded and randomized, there was no difficulty indistinguishing between the specimens from the drug-treated and thesaline treated animals. The most prolific nerve growth occurred in theanimals treated with c(PG)3.

Nerve regeneration is promoted by thoroughly bathing the injury sitewith the foregoing composition. The foregoing composition promotesregeneration of damaged nerve tissue when administered to the site ofthe injury, including direct administration to the site of injury.

Experiment 11

Regeneration of Damaged Nerve Tissue of Cyclic Glycyl-2-Methyl Proline

Postnatal day 4 Wistar 10 rats were used for the study. The rats weredivided in 2 groups: one group of 5 animals treated with drug solution,and the other group of 5 animals treated with 0.9% sodium chloride(saline) solution. A drug solution containing 10 mM of cyclicGlycyl-2-Methyl Proline, or cGMeP (from NeuroBiomed, 10 mM solutionprepared in 0.9% sodium chloride (saline) solution) was administeredfour times a day in volumes of 0.75 ml to thoroughly flood the site ofinjury.

Two days after crushing the spinal cord, the dura of a rat was openedand a polyethylene tube was sutured to the vertebral spines and adjacentsoft tissues so that the opening in one end lay directly over theinjured part of the spinal cord. The tubing was brought through asubcutaneous tunnel so that its other end emerged at the base of theskull. A syringe adapter was attached to the external opening forinjecting the drugs. All experiments were on a double-blind basis on 2groups: one group of 5 animals treated with drug solution of 10 mM cGMePin saline solution, and the other group of 5 animals treated with 0.9%sodium chloride (saline) solution. Treatment of every animal wascontinued for 14 days, after which the animals were killed andhistological sections prepared.

Results

The results are similar to the above experiment with cPG. In the cGMePtreated animals, the nerve fibers grew into the lesion site in suchprofusion that they were no longer oriented longitudinally, but grewrather haphazardly in all directions. Fibers were frequently undulatingand varicose and were often arranged in small bundles containing 3-6axons. The axons were very fine in caliber, most of them being 3-7microns in diameter.

When the slides were coded and randomized, there was no difficulty indistinguishing between the specimens from the drug-treated and thesaline treated animals. The most prolific nerve growth occurred in theanimals treated with cGMeP.

Nerve regeneration is promoted by contacting, such as but limited tobathing the injury site with the foregoing composition. The foregoingcomposition promotes regeneration of damaged nerve tissue whenadministered directly to the site of the injury.

Experiment 12

Clinical Trial on Alzheimer's Patients with Mild Cognitive Impairment.

Mild cognitive impairment (“MCI”) is a syndrome defined as cognitivedecline greater than expected for an individual's age and educationlevel. Mild cognitive impairments are those involving impairments ofmemory and other cognition functions, beyond the age norm but notleading to the characteristic of dementia.

Mild cognitive impairment in population-based studies ranges from 3% to15% in adults older than 65 years. More than half progress of peoplewith MCI progress to dementia within 5 years.

The detection of mild cognitive disorders is important, becausetreatment can have the greatest efficacy at this stage than afterdementia has developed.

Materials and Methods

A randomized clinical trial of NA-831 (also known as cyclic ProlylGlycine) was performed on Alzheimer patients with mild cognitiveimpairment of vascular origin. The drag NA-831 was administered orallywith a one capsule of 10 mg once a day over a period of 12 weeks. Therewere 32 Alzheimer patients participated in the study.

Inclusion Criteria

-   -   Is male or female, at 55-85 years of age (inclusive) at        screening.    -   Self-reported memory complaint, corroborated by spouse or        companion as appropriate.    -   Wechsler Memory Scale III (WMS-III) age-adjusted Logical Memory        II score ≤5.    -   Mini-Mental State Exam (MMSE)≥24.    -   Center for Epidemiologic Studies-Depression (CES-D) score <27.    -   Normal thyroid function, defined as TSH, T3 and T4 within normal        limits.    -   Agree not to consume alcoholic beverages within 8 hours of each        study visit.    -   Willing and able to sign informed consent and complete the CTB        and all other tests and        -   procedures as listed in the protocol.    -   Female subjects must be surgically sterile or post-menopausal        for at least 2 years. If <2 years post-menopausal, then a        follicle stimulating hormone (FSH) ≥40 mlU/mL must be obtained.

Exclusion Criteria:

-   -   Subjects who have any significant, untreated psychiatric illness        or any CNS condition (such as schizophrenia, Parkinson's        disease, stroke, etc.) that could interfere with the study        evaluations or procedures or which poses an additional risk.    -   Subjects with a history of uncomplicated depression may        participate if in remission and on a stable dose of        antidepressant medication for at least 2 months.    -   History of significant head trauma followed by persistent        neurologic defaults or known structural brain abnormalities.    -   Have had a stroke or Transient Ischemic Attack (TIA) or        unexplained loss of consciousness in the past 1 year    -   History of unstable angina, myocardial infarction, chronic heart        failure or clinically significant conduction abnormalities        within 1 year prior to Screening Visit 1    -   History of alcohol or substance abuse or dependence within the        past year.    -   Acute infective sinusitis.    -   History or presence of an abnormality of the external or        internal structures of the nose or nasopharynx, except for        surgical correction of the nasal septum or a “broken nose” at        least 2 years previously, or surgical repair of cleft palate        when <30 years of age.    -   Use of medications that are known to cause frank obtundation of        cognition    -   Use of any approved or investigational medication for        Alzheimer's Disease within 3 months of screening    -   History of or current significant systemic disease judged to        interfere with the study evaluations or likely to be a safety        concern.    -   Untreated sleep apnea or treatment for sleep apnea for <3        months.    -   Clinically significant systemic illness or serious infection        within 30 days prior to or during the screening period    -   Use of allowed medications for chronic conditions at doses that        have not been stable for at least 4 weeks prior to Screening        Visit 1, or use of AD medications at doses that have not been        stable for at least 8 weeks prior to Screening Visit 1.    -   Abnormal clinical laboratory test results, specifically: Alanine        transaminase (ALT) or aspartate transaminase (AST)>2× the upper        limit of normal (ULN), Hematology <80% the lower limit of        normal, Creatinine ≥2 mg/dL and other clinical laboratory values        or vital signs considered clinically significant in the opinion        of the Investigator.    -   Treatment with any investigational drug, biologic, or device        within the previous 30 days prior to screening.    -   Surgery involving general anaesthesia within the past 3 months        or planned surgery requiring general anaesthesia during the        study period.    -   Contraindications to study procedures    -   Use of my medications that, in the opinion of the Investigator,        may contribute to cognitive impairment, put the participants at        higher risk for adverse events (AEs), or impair the        participant's ability to perform cognitive testing or complete        study procedures.

Assessment

-   -   1) A scale assessing the severity of symptoms based on the        Unified Assessment of Clinical-Pharmacological Actions of        Psychotropic Agents in Patients with Organic Disorders, which        provides objective quantitative data on the therapeutic dynamics        of psychopathological symptomatology and the characteristics of        the psychotropic actions of agents.    -   2) The Mini Mental State Examination (MMSE), which consists of a        battery of neuropsychological tests evaluating cognitive        functions in points: attention, memory, gnosis, speech, praxis,        and counting.    -   3) The Brief Cognitive Rating Scale (BCRS), which provides        assessment of the severities of the individual components of        cognitive Impairments.    -   4) The Cognitive Capacity Screening Examination (CCSE), which        consists of a battery of tests assessing cognitive functions:        orientation, memory, counting, and the abilities to infer and to        group objects.    -   5) The Clinical Global Impression scale, which provides        quantitative assessments of the therapeutic efficacies of        agents, along with tolerance and safety, using investigations of        measures of disease severity, the level of “overall        improvement,” the therapeutic effect, and the presence and        severity of side effects during treatment.    -   6) Laboratory analyses consisting of general blood and urine        tests, including biochemical investigations (AST, ALT).    -   7) ECG traces.

Results

The mental state of patients with mild cognitive impairment, such as butnot limited to through vascular origin was defined by the assessmentincluding the Mini Mental State Examination (MMSE), the Brief CognitiveRating Scale (BCRS), the Cognitive Capacity Screening Examination(CCSE).

The therapeutic actions of NA-831 in the patients includes decreases inneurosis-like symptomatology and cognitive disorders.

The results are given in Table 2:

TABLE 2 Mild Cognitive Impairment Measurement Data Mild Co

itive Impairment - Clinical Data Baseline  Baseline  Baseline  Baseline Baseline  Baseline  Baseline day 7  day 14  day 21  day 28  day 42  day56  day 70 Measure W p W p W p W p W p W p W p Increased fatigue17 >0.05 95 0.02 97 0.02 137 0.01 155 0 159 0 162 0.001 Anxiety 22 >0.0522 >0.05 46 0.02 70 0.02 107 0 110 0 115 0.001 Decreased mood 3 >0.0512 >0.05 22 0.03 23 0.03 23 0 23 0 24 0.03 Apathy indifference 19  .0.0541 0.02 57 0.02 59 0.02 61 0 65 0 67 0.02 Increased irritability 38  0.02 47 0.02 68 0.01 93 0 109 0 110 0 117 0.001 Affective Lability 23  0.03 58 0.02 69 0.01 118 0.01 139 0 140 0 142 0.001 SleepingDisturbance 37   0.03 47 0.02 70 0.02 70 0.02 82 0 82 0 82 0.01Disturbance to waking 21 >0.05 29 0.02 41 0.02 47 0.02 69 0 92 0 950.001 Daytime drowsiness 19 >0.05 45 0.02 52 0.02 67 0.01 76 0 109 0 1170.001 Headache 28   0.02 49 0.02 98 0 101 0 120 0 121 0 129 0.001Orthostatic Impairment 18 >0.05 37 0.02 58 0.02 78 0.01 67 0 78 0 810.02 Tachycardia 29   0.02 28 32 35 0.02 45 0.02 53 0 67 0 69 0.01Hyperesthesia 11 >0.05 21 0.03 33 0.04 67 0.01 69 0 78 0 83 0.01Sweating 12 >0.05 23 >0.05 37 0.02 66 0.01 67 0 67 0 69 0.01 Impairmentsto the 37   0.02 57 0.02 91 0 93 0 104 0 113 0 118 0.001 depth andduration of nocturnal sleep

indicates data missing or illegible when filed

W is Wilcoxon signed-rank test, which is the nonparametric testequivalent to the dependent t-test. It is used to compare two sets ofscores that come from the same participants. This can occur when we wishto investigate any change in scores from one time point to another, orwhen individuals are subjected to more than one condition.

Clinicians use used a Wilcoxon signed-rank test to understand whetherthere was a difference in the patient conditions in term of fatiguebefore and after a period the time with the drag treatment.

Note that NA-831 has been shown to significantly improve the followingfunctions with the p<0.01:

-   -   Reduce fatigue    -   Reduce anxiety    -   Reduce irritability    -   Improve lability    -   Reduce disturbance from waking up at night    -   Reduce daytime drowsiness    -   Improve duration of nocturnal sleep

Analysis of the therapeutic efficacies of NA-831 were performed in allpatients in the trial using the assessment methods including: the MiniMental State Examination (MMSE), the Brief Cognitive Rating Scale(BCRS), and the Cognitive Capacity Screening Examination (CCSE).

The clinical data on the efficacy of NA-831 in Alzheimer patents can besummarized and highlighted as follows:

NA-831 was found to improve the patient's ability to concentrate andcount. In the graph, the Measurement of Brief Cognitive Rating Scale wasplotted against time, the statistical data after day 42 to day 84, withp value of less than 0.01. This means that they have significantlypositive impacts in improving patients' concentration and ability tocount. See, FIG. 9.

It is a common symptom that Alzheimer patients' short-term memorybecomes progressively worse. NA-831 not only improves the short-termmemory, but also long-term memory. See, FIG. 10.

In addition, Alzheimer patients' long term memory becomes progressivelyworse. NA-831 not only improves the short-term memory, but alsolong-term memory. See, FIG. 11.

In the early stages of the disease, Alzheimer patients often becomedisoriented even in their oven home. NA-831 improves the orientation andrestore their sense of time and place. See, FIG. 12.

As the disease progresses, Alzheimer patients cannot take care ofthemselves. The drag improves patient's ability to take care of theirdaily activity and self-care. See, FIG. 13.

The Mini-Mental State Examination (MMSE) is a 30-point questionnairethat is used extensively in clinical and research settings to measurecognitive impairment, the impact of NA-831 on the emotion of thepatients were shown with a significant improvement in the MMSE from anaverage of 23.5 (mild impairment) on day 1 to 29.75 (normal) on day 84.See, FIG. 14.

NA-831 was found to have high efficacy in the treatment of patients withmild cognitive impairment with marked improvement in 92.5% of allpatient, and little improvement in 7.5% of patients. See, FIG. 15.

Conclusions

Cyclic Prolyl Glycine decreased or prevented glutamate-inducedneurotoxicity, indicating that the drug is neuroprotective and can beused to inhibit neuronal degeneration or cell death.

Experiment 13

Assay to Study the Relationship of ePG and NB2B-Subfype Receptors inAnimals after Intravenous Administration.

In this experiment 8-10 weeks old male CD-1 mice (n=12) wereadministered intravenously in a vehicle consisting of 10%dimethylacetamide, 40% PEG-400, 30% hydroxypropyl betacyclodextrin, and30% water with cyclic Prolyl Glycine (0.10 mg/ml), and the forebrainswere harvested 15 minutes post-dosing by decapitation. The brain sampleswere immediately frozen and stored at −80° C.

On the following day, the dosed brain samples were thawed on ice for20-30 minutes followed by homogenization using Polytron for 10 secondsin cold homogenization buffer composed of 50 mM KH₂PO₄ (pH adjusted to7.4 with KOH), 1 mM EDTA, 0.005% Triton X 100 and protease inhibitorcocktail (Fischer Scientific). The crude homogenates were furtherhomogenized using a Bounce homogenizer (Thomas Scientific) and thehomogenized membrane aliquots from all animals were gash-frozen andstored at ˜80° C. until further use. The whole homogenization processwas performed on ice.

For determining occupancy, the membrane homogenates were thawed on iceand then needle-homogenized using a 25-gauge needle. The homogenizedmembrane (6.4 mg/ml) was added to a 96-well plate followed by additionof ³H Ro 25-6981 (6 nM). The reaction mixture was incubated for 5minutes on a shaker at 4° C. and then harvested onto GF/B alter plates(treated with 0.5% PEI for 1 hour at room temperature). The alter plateswere dried at 50° C. for 30 mins, incubated with microscintillation 20for 15 minutes and read by a benchtop microplate scintillation andluminescence counter (TopCount Model NXT manufactured by Perkin Elmer).Each dose or compound group consisted of 4 animals. The control group ofanimals was dosed with vehicle alone. Membrane from each animal wasadded in triplicates to the assay plate. Non-specific binding wasdetermined using 10 μM Ro 25-6981 added to the wells containing membranehomogenates from vehicle-dosed animals.

Specific counts/minute was converted to % occupancy at each dose of acompound for each animal using the following equation:

${\%\mspace{14mu}{Occupancy}\mspace{14mu}\left( {{animal}\mspace{14mu} A} \right)} = {{\frac{{specific}\mspace{14mu}{CPM}\mspace{14mu}{of}\mspace{14mu}{animal}\mspace{14mu} A}{{Specific}\mspace{14mu}{CPM}\mspace{20mu}{from}\mspace{14mu}{control}\mspace{14mu}{group}}\mspace{11mu} 100} - \left( {\times 100} \right)}$

Using this procedure, cPG compound showed 93% NR2B receptor occupancyafter a 3 mg/Kg i.v. dose. Drug levels were determined by massspectroscopy method. Drug levels in the blood plasma were 1074 nM in atthis dose, and drug levels in the homogenized brain tissue were 1632 nM.

The results indicate that cPG occupies brain-resident NR2B-subtypereceptors in animals after intravenous administration and that thecompound and its analogues are pharmaceutically effective.

Experiment 14

Mouse Forced Swim Test (mFST).

Forced Swim Test (FST) is an animal model used to assess antidepressantcompounds in preclinical studies. The FST was performed similar to themethod of Porsolt et al. with modifications (Porsolt R D, Bertin A,Jalfre M. Behavioral despair in mice: a primary screening test forantidepressants. Arch Int Pharmacodyn Thér 1977; 229:327-36).

In this experiment, mice are forced to swim in an inescapable cylinderfilled with water. Under these conditions, mice will initially try toescape and eventually develop immobility behavior; this behavior isinterpreted as a passive stress-coping strategy or depression-likebehavior. Swim tanks were positioned inside a box made of plastic. Eachtank was separated from each other by opaque plastic sheets to theheight of cylinders. Three mice were subjected to test at a time. Swimsessions were conducted for 6 min by placing mice in individual glasscylinders (46 cm height×20 cm diameter) containing water (20-cm deep,maintained at 24-25° C.). At this water level, the mouse tail does nottouch the bottom of the container. The mouse was judged to be immobilewhenever it remained floating passively without straggling in the waterand only making those movements necessary to keep its nose/head abovethe water and to keep it afloat. The duration of immobility wasevaluated during the total 6 min of the test and expressed as duration(sec) of immobility. Each mouse was tested only once. At the end of eachsession, mice were dried with a dry cloth and returned to their homecage placed on a thermal blanket to prevent hypothermia. Water wasreplaced after each trial.

The results obtained in the FST were shown as an arithmetic mean ofimmobility time of animals (given in seconds)±standard error of the mean(SEM) for each experimental group.

In order to avoid the risk of obtaining the false positive/negativeeffects in the FST caused by a possible influence of the tested drugs onthe locomotor activity, spontaneous locomotor activity was measuredusing an animal activity meter Opto-Varimex-4 Auto-Track (ColumbusInstruments, USA). The device consists of four transparent cages with alid (43×43×32 cm), a set of four infrared emitters (each emitter has 16laser beams), and four detectors monitoring animal movements. Each mousewas placed individually into the cage for 10 min. Spontaneous locomotoractivity was evaluated between the 2^(nd) and the 6^(th) min, whichcorresponds with the time interval analysed in the FST.

In addition, all testing sessions were recorded with a video camera(Sony Handicam, Model: DCR-HC38E; PAL) and scoring was done using theForced Swim Scan, Version 2.0 software (Clever Systems Inc., Reston,Va., USA; see Hayashi E, Shimamura M, Kuratani K, Kinoshita M, Hara H.Automated experimental system capturing three behavioral componentsduring murine forced swim test. Life Sci. 2011 Feb. 28; 88(9-10):411-7and Yuan P, Tragon T, Xia M, Leclair C A, Skoumbourdis A P, Zheng W,Thomas C J, Huang R, Austin C P, Chen G, Guitart X. Phosphodiesterase 4inhibitors enhance sexual pleasure-seeking activity in rodents.Pharmacol Biochem Behav. 2011; 98(3):349-55).

Cyclic Prolyl Glycine (at concentration 0.10 mg/ml) was administered in12 mice thirty minutes before swim session by i.v. route and immobilitytime was recorded for next 6 min. AY the end of FST, the mouse waseuthanized by rapid decapitation method and plasma and brain sampleswere collected and stored under −80° C. till further analysis. In themouse forced swim assay, the cPG compound was dosed intravenously in avehicle of saline solution (0.90% Sodium Chloride) at a 5 mL/Kg dosingvolume. The compound demonstrated a statistically significant decreasein immobility time at 1 mg/Kg under these conditions. Drug levels were237+/−128 nM in the plasma and 632+/−173 nM in the brain at this dose.The NR2B receptor occupancy was determined as reported above and wasdetermined to be 73%. The cPG analog demonstrated a statisticallysignificant decrease in immobility time at 1 mg/Kg under these sameconditions. Drug levels were 215 nM in the plasma. The NR2B receptoroccupancy was determined to be 68%.

The results indicate that cyclic Prolyl Glycine (NA-831) compoundsexhibit antidepressant properties.

Experiment 15

Clinical Study: A Double-Blind, Randomized, Placebo-Controlled, ActiveReference Study of Cyclic Prolyl Glycine (NA-831) in Patients with MajorDepressive Disorder.

Introduction

Cyclic Prolyl Glycine (NA-831) is a novel compound under development asan antidepressant. Based on preclinical data, these affinities areconsidered to be of clinical relevance and involved in the mechanism ofaction at therapeutic doses. In vivo, NA-831 increases the extracellularlevels of serotonin (5-HT), noradrenaline, dopamine, acetylcholine andhistamine in rat prefrontal cortex and hippocampus. The aim of thisclinical study was to investigate the efficacy, safety, and tolerabilityof two fixed doses (20 and 40 mg/d) of NA-831 vs. that of placebo after6 week treatment in adult patients with major depressive disorder (MDD),Venlafaxine XR was used as the active reference.

Method

This randomized, double-blind, fixed-dose, placebo-controlled, activereference study recruited 32 randomized patients in accordance with theprinciples of Good Clinical Practice [ICH (1996). Harmonized TripartiteGuideline E6: Guideline for Good Clinical Practice(http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm073122.pdf)] and the Declaration of Helsinki [WMA (1964).Ethical Principles for Medical Research Involving Human Subjects(http://www.wma.net/en/30publications/10policies/b3/). World MedicalAssociation.] Local ethics committees approved the study design andeligible patients gave their written informed consent beforeparticipating.

Eligible patients were randomized equally (1:1:1:1) to one of the fourtreatment arms for a 6-week double-blind treatment period. Randomizedpatients were given 1-week wallet cards at each visit and wereinstructed to take two capsules per day, orally, at the same time everyday (preferably in the morning). NA-831 was dosed at 20 mg/day or 40mg/d for 6 week and venlafaxine at 75 mg/d over 6-week treatment period.Efficacy and tolerability were assessed at screening, baseline and after1, 2, 3, 4, 5, and 6 week. Patients were contacted for a safetyfollow-up 4 week after the completion visit.

Main Entry Criteria

Patients with MDD presenting with a current major depressive episodeaccording to DSM-IV-TR criteria were included in the study if they werean outpatient of either sex, aged from 20 yr to 65 yr, (mean=39.7±8.5)(with a Montgomery-Åsberg Depression Rating Scale (MADRS) (Montgomery &Åsberg, 1979) total score≥30 at the baseline visit. [Montgomery S AsbergM (1979). A new depression scale designed to be sensitive to change.British Journal of Psychiatry 134,382-389.https://doi.org/10.1192/bjp.134.4.382]

Patients were excluded if they had any current psychiatric disorderother than MOD as defined in Diagnostic and Statistical Manual of MentalDisorders, 4^(th) Edition (DSM-IV-TR), assessed using the MiniInternational Neuropsychiatric Interview [Sheehan D V Lecrubier YSheenan K H Amorim P et al. (1998). The Mini-InternationalNeuropsychiatric Interview (M.I.N.I.). Journal of Clinical Psychiatry 59(Suppl. 20), 22-33, quiz 34-57] or if they had a current or past historyof manic or hypomanic episode, schizophrenia or any other psychoticdisorder, including major depression with psychotic features, mentalretardation, organic mental disorders, or mental disorders due to ageneral medical condition, any substance abuse disorder within theprevious 6 months, presence or history of a clinically significantneurological disorder (including epilepsy), any neurodegenerativedisorder, or any Axis II disorder that might compromise the study.

Patients at serious risk of suicide, based on the investigator'sclinical judgement, or who had a score of ≥5 on item 10 of the MADRSscale (suicidal thoughts) were also excluded, as were those receivingformal behaviour therapy or systematic psychotherapy, or were pregnantor breastfeeding, had a known hypersensitivity or were non-response tovenlafaxine, or whose current depressive symptoms were considered by theinvestigator to have been resistant to two adequate antidepressanttreatments of at least 6 week duration, or had previously been exposedto NA-831.

Patients were also excluded if they were taking the followingpsychotropic drugs within 2 week prior to baseline or during the study:Reversible or irreversible monoamine oxidase inhibitors, SSRIs(fluoxetine within 5 week), SNRIs, tricyclic antidepressants,psychoactive herbal remedies, any drug used for augmentation ofantidepressant action or any other antidepressant drugs, oralantipsychotic and anti-manic drugs, or dopamine ants, any anxiolytics(including benzodiazepines); and any anticonvulsant drug, serotonergics, narcotic analgesics or cough agents, anti-arrhythmics, oralanticoagulants, proton pump inhibitors, steroids, cisapride, macrolideantibiotics, antifungal agents, antihypertensives, all anti-inflammatoryagents, anti-migraine agents, pseudoephedrine, hypolipidaemics, andepisodic use of insulin. Occasional use of zolpidem, zopiclone andzaleplon for insomnia was allowed.

Patients were withdrawn if they became pregnant during the study, if theinvestigator considered it to be in the best interest of the patient forsafety/efficacy reasons, if laboratory values were outside normal rangesand clinically significant, if they were considered to be at significantrisk of suicide, if they scored ≥5 points on item 10 (suicidal thoughts)of the MADRS, if the randomization code for a patient was broken, ifconsent to participate was withdrawn, if they did not take studymedication for more than 6 consecutive days, or if the patient was lostto follow-up. The patient could be withdrawn from the study if a seriousadverse event (SAE) occurred. If adverse events (AEs) were contributoryto withdrawal, they were always regarded as the primary reason forwithdrawal.

Efficacy Rating

Patients were evaluated using the MADRS from baseline to week 6. Ratertraining was undertaken to increase inter-rater reliability, and wassupervised by an experienced investigator. Only those investigators whohad actively participated in rater training sessions prior to inclusionof patients into the study were allowed to rate patients. Patientratings were assessed by the same investigator at each visit, wheneverpossible.

Allocation to Treatment

The medication was given as capsules of identical appearance. Patientswho met the selection criteria at the baseline visit were assigned todouble-blind treatment according to a computer-generated randomizationlist. The details of the randomization series were unknown to any of theinvestigators and were contained in a set of sealed opaque envelopes. Ateach study site, sequentially enrolled patients were assigned the lowestrandomization number available in blocks of four. Ail investigators,study personnel and participants were blinded to treatment assignmentfor the duration of the entire study.

Analysis Sets

All safety analyses were based on the all-patients-treated set (APTS),comprising all randomized patients who took at least one dose of studymedication. All efficacy analyses were based on a modifiedintent-to-treat set (ITT)—the full-analysis set (FAS), comprising allpatients in the APTS who had at least one valid post-baseline MADRStotal score assessment.

Primary Efficacy Analysis

Four hypotheses were part of the primary efficacy analysis, which wasfully adjusted for multiplicity using a hierarchical testing procedureat the 5% level of significance as long as the previous hypothesis wasrejected. The order of testing was: no difference between the 20 mg dosevs. placebo at week 6, no difference between 40 mg vs. placebo at week6, no difference between 20 mg dose vs. placebo at week 1, and finallyno difference between 40 mg dose vs. placebo at week 1. The statisticalmodel was an analysis of covariance (ANCOVA) of the change from baselinein MADRS total score (FAS, LOCF) with treatment and site as fixedfactors and the baseline MADRS score as a covariate. The primaryefficacy analysis was repeated on observed cases (OC) data, using bothan ANCOVA and a mixed model for repeated measurements (MMRM).

Tolerability Assessments

All adverse effects (AEs) (including any change in concurrent illnessesor new illnesses) either observed by the investigator or reportedspontaneously by the patient were recorded. AEs were coded using thelowest level term according to the Medical Dictionary for RegulatoryActivities, version 10.0. As a post-hoc analysis, the safety databasewas searched at preferred-term and verbatim-term level for possiblesuicide-related AEs, as described by the FDA [Laughren T (2006).Memorandum on Suicidality.(http://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4272b1-01-fda.pdf)].

Results

Patient Baseline Characteristics

The APTS comprised 32 patients (placebo, 8, venlafaxine, 8; 20 mgNA-831, 8, 40 mg NA-831,8). There were no clinically relevant orstatistically significant differences between the treatment groups inpatient demographics or clinical characteristics at baseline (Table 3).Patients had a mean age (±S.D.) of 39.7±8.5 yr, 59.4% were women. Themean baseline MADRS total score was 34.0, indicating a severelydepressed patient population, consistent with the mean CGI-S score of5.1. Patients were diagnosed with their first MDE˜10 yr prior toenrolment. Between 74% and 80% of the patients in each treatment grouphad had a previous MDE and their current episode had started about 5months prior to enrolment.

TABLE 3 Baseline patient characteristics NA-831 NA-831 VenlafaxinePlacebo (n = 8) 20 mg (n = 8) 40 mg (n = 8) 225 mg (n = 8) Women 5(62.5% 5 (62.5%) 5 (50.0 %) 5 (62.5 %) Age (yr) Mean ± SD 40.0 ± 10.741.3 ± 8.9 39.6 ± 10.4 38.0 ± 10.3 Range 21-61 21-62 19-60 20-63Patients with first MDE 21.50% 22.50% 23.00% 22.50% Years since first  10 ± 4   10 ± 3   9 ± 3   11 ± 9 MDE ± SD. Days since start of  165 ±34  161 ± 39  163 ± 35 160 ± 47 current MDE ± SD Efficacy scores (n =105) (n = 108) (n = 100) (n = 112) MADRS total score ± SD 33.4 ± 2.734.1 ± 2.6 34.0 ± 2.8 33.2 ± 3.1 Based on the full-analysis set : CGI-S,Clinical Global Impression- Severity; MADRS: Montgomery-ÅsbergDepression Rating Scale; MDE: major depressive episode; S.D.: standarddeviation.Withdrawals from the Study

Only 4 subjects withdrew because of various reason: one from the placebogroup, one from the 40 mg NA-831 group and 2 from the Venlafaxine group.More than 87% of the patients completed the study.

Efficacy

On the pre-defined primary efficacy endpoint, both doses of NA-831 werestatistically significantly (p<0.0001) superior to placebo in meanchange from baseline m MADRS total score at week 6 (FAS, LOCF), withmean treatment differences to placebo of 7.7 (20 mg) and 8.5 (40 mg)points (Table 4) in a multiplicity-controlled analysis. Venlafaxine wasalso statistically significantly (p<0.0001) superior to placebo at week6, with a mean treatment difference to placebo of 7.2 points (LOCF). Theestimated treatment differences and nominal p values at week 6 obtainedfrom an analysis using MMRM were similar to those obtained in the ANCOVAanalyses 5.7±1.3 (20 mg NA-831), 7.8±1.3 (40 mg NA-831), 5.6±1.3(venlafaxine), all p<0.0001] (Table 2),

TABLE 4 Summary of Efficacy Analysis Difference to Analysis TreatmentGroup Mean ± SD Placebo p value LOCF, Placebo (n = 7) −14.7 ± 1.0 — —ANCOVA NA-831 20 mg (n = 8) −22.4 ± 1.0 −7.7 ± 1.4 <0.0001 NA-831 40 mg(n = 7) −23.2 ± 1.0 −8.5 ± 1.4 <0.0001 Venlafaxine (n = 6) −21.9 ± 1.0−7.7 ± 1.4 <0.0001 OC, Placebo (n = 7) −16.6 ± 1.0 — — ANCOVA NA-831 20mg (n = 8) −22.3 ± 0.9 −5.7 ± 1.3 <0.0001 NA-831 40 mg (n = 7) −24.4 ±1.0 −7.8 ± 1.3 <0.0001 Venlafaxine (n = 6) −22.2 ± 0.9 −5.6 ± 1.3<0.0001 MMRM Placebo (n = 7) −15.4 ± 1.0 — — NA-831 20 mg (n = 8) −22.3± 0.9 −6.9 ± 1.3 <0.0001 NA-831 40 mg (n = 7) −24.9 ± 1.1 −9.5 ± 1.4<0.0001 Venlafaxine (n = 6) −23.5 ± 0.9 −8.1 ± 1.3 <0.0001 FAS:Full-analysis set LOCF: last observation carried forward MADRS:Montgomery-Åsberg Depression Rating Scale; MMRM: mixed model repeatedmeasures; OC: observed cases; S.E:, standard error of the mean.

Tolerability and Safety

During the 6-week treatment period, approximately 60% of patients in theplacebo group and 75% of the venlafaxine groups had one or more AE. Only12.5% of 20 mg NA-831 groups and 12.5% of the patients in the 40 mgNA-83 had AEs.

A total of 10 (33.3%) patients withdrew due to AEs: 1 (10%) in theplacebo group, no withdrawal from the 20 mg NA-831 group, 1 (12.5%) inthe 20 mg NA-831 group, and 2 (25%) in the venlafaxine group.

The most common AEs reported in the active NA-831 treatment groups weremild headache and dry mouth. A majority of patients in the venlafaxinegroup reported nausea, severe headache loss of strength, blurred vision,chest pain, fast or irregular heartbeat and suicidal thoughts.

Conclusion

The aim of the double-blind, randomized, placebo-controlled study was toevaluate the efficacy, safety and tolerability of NA-831 in patientswith MDD. The active reference, venlafaxine XR was included with thepurpose of validating the study methodology and patient population, andwas effective on the primary efficacy analysis. Both doses of NA-831resulted in a significant improvement compared to placebo on the primaryefficacy analysis.

The difference between active treatment and placebo of ˜7 points on theMADRS translates into a clinically relevant difference in response ratesof 32.5% units, compared to an average of 16% units for antidepressantsapproved by the European authorities [Melander H Salmonson T Abadie Evan Zwieten-Boot B (2008). A regulatory apologia—A review ofplacebo-controlled studies in regulatory submissions of new-generationantidepressants. European Neuropsychopharmacology 18,623-627.https://doi.org/10.1016/j.euroneuro.2008.06.003]

In conclusion, treatment with NA-831 for 6 week in this study was welltolerated and efficacious in reducing depressive and anxious symptoms inpatients with MDD.

The present invention is described with reference to specificembodiments thereof. Other features and embodiments of this inventioncan be produced by those of skill in the art without undueexperimentation and a reasonably likelihood of success. All of those andother embodiments are considered to be part of this invention.

Advantages of the Present Invention

Some advantages offered by the present invention with the cyclicpeptides, especially over IGF-1 include:

The active ingredients are easy to synthesize either in vitro or byother means such as recombinant techniques.

The peptide as a small molecule can diffuse readily through the body andbetween compartments (e.g. the blood-brain barrier, and mucousmembranes), aiding in the choice of methods for its administration andits ability to reach sites where injury has occurred.

cPG, c(PG)3 and cGMeP are very stable molecule and is unlikely topresent a challenge to the immune system, so it may be administered overextended periods and it may be administered prophylactically.

The present invention provides a novel therapeutic method for preventingbrain injury and degenerative diseases by regulating mGluRs particularly⅔ leading to long-term benefits of brain recovery.

With a role in regulating IGF-1 induction, cPG will provide furtherneuroprotection with less potential for growth side-effects.

Conclusions

Cyclic Prolyl Glycine decreased or prevented glutamate-inducedneurotoxicity, indicating that the drug Is neuroprotective and can beused to inhibit neuronal degeneration or cell death.

The present invention is described with reference to specificembodiments thereof. Other features and embodiments of this inventioncan be produced by those of skill in the art without undueexperimentation and a reasonably likelihood of success. All of those andother embodiments are considered to be part of this invention.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexamples only, and not limitation. It will be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the spirit and scope of the presentinvention as defined in the appended claims. Thus, the breadth and scopeof the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined inaccordance with the following claims and their equivalents.

All publication, including patent documents and scientific articles,referred to in this application, including any bibliography, areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication were individually Incorporatedby reference. All headings are for the convenience of the reader andshould not be used to limit the meaning of the text that follows theheading, unless so specified.

What is claimed is:
 1. A method of preventing the symptoms ofParkinson's disease in a mammal, comprising: a. providing a mammal inneed of preventing the symptoms of Parkinson's disease; b. administeringa pharmaceutically effective amount of cyclic Prolyl Glycine (cPG) orits analogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl 2-AllylProline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), or a combination thereof, collectivelycalled a cPG compound, to said mammal; wherein said mammal is preventedfrom having the symptoms of Parkinson's disease.
 2. The method accordingto claim 1, wherein said pharmaceutically effective amount of said cPGcompound is from about 1 μg to about 100 mg per kg of body weight. 3.The method of claim 1, wherein said pharmaceutically effective amount ofsaid cPG compound is administered at an amount from about 0.1 mg toabout 10 mg/kg per day, from about 0.5 mg to about 20 mg/kg per day,from about 0.2 mg to about 40 mg/kg per day, from about 5 mg to about 50mg/kg per day, or from about 10 micrograms to about 100 mg/kg per day.4. The method of claim 1, wherein said pharmaceutically effective amountor said cPG compound has a lower limit of about 0.1 milligrams perkilogram mass (mg/kg) of said mammal and an upper limit of about 10mg/kg of said mammal.
 5. The method of claim 1 wherein saidpharmaceutically effective amount of said cPG compound is between about20 mg and about 80 mg per day, or between about 20 mg and about 100 perday.
 6. The method according to claim 1, wherein said administering isin the form of a pharmaceutical composition including a pharmaceuticallyacceptable carrier.
 7. The method according to claim 1, wherein saidadministering is in combination with artificial cerebrospinal fluid. 8.The method according to claim 1, wherein said administering isintravenous.
 9. The method according to claim 1, wherein saidadministering is combined with the administration of a neuroprotectiveagent, insulin-like growth factor-I (IGF-I) insulin growth-dikefactor-II (IGF-II), or a combination thereof.
 10. The method accordingto claim 1, wherein said administering is combined with theadministration of an anti-inflammatory agent, an anti-integrin alpha 4subunit rea gent, or a combination thereof.
 11. The method of claim 1,wherein said administering is combined with administration of ananti-inflammatory agent.
 12. The method of claim 1, wherein said cPGcompound comprises an aqueous solution and one or more pharmaceuticallyacceptable excipients, additives, carriers or adjuvants.
 13. The methodof claim 1, wherein said ePG compound comprises one or morepharmaceutically acceptable excipients, carriers, additives, adjuvantsor binders in a tablet or capsule.
 14. The method of claim 1, whereinsaid pharmaceutically effective amount of said cPG compound isadministered via an oral, intraperitoneal, intravascular, peripheralcirculation, subcutaneous, intraorbital, ophthalmic, intraspinal,intracisternal, topical, infusion, implant, aerosol, inhalation,scarification, intracapsular, intramuscular, intranasal, buccal,transdermal, pulmonary, rectal, vaginal, or a combination thereof. 15.The method of claim 1, wherein said mammal is a human.
 16. A method oftreating the symptoms of Parkinson's disease in a mammal, comprising: a.providing a mammal in need of treating of the symptoms of Parkinson'sdisease; b. administering a pharmaceutically effective amount of cyclicProlyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine) orcyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline or cyclicGlycyl-2-Methyl-Proline (cPMeG), or a combination thereof, collectivelycalled a cPG compound, to said mammal; wherein said mammal is treatedfor symptoms of Parkinson's disease.
 17. A method of preventing thesymptoms of Dementia with Lewy Bodies in a mammal, comprising: a.providing a mammal in need of preventing the symptoms of Dementia withLewy Bodies; b. administering a pharmaceutically effective amount ofcyclic Prolyl Glycine (cPG) or its analogues (cyclic(tri(Prolyl Glycine)or cyclic Glycyl-2-Allyl Proline, or cyclic Glycyl-Alkyl Proline orcyclic Glycyl-2-Methyl-Proline (cPMeG), or a combination thereof,collectively called a cPG compound, to said mammal; wherein said mammalis prevented from having the symptoms of Dementia with Lewy bodies. 18.A method of treating the symptoms of Dementia with Lewy Bodies in amammal, comprising: a. providing a mammal in need of treating thesymptoms of Dementia with Lewy Bodies; b. administering apharmaceutically effective amount of cyclic Prolyl Glycine (cPG) or itsanalogues (cyclic(tri(Prolyl Glycine) or cyclic Glycyl-2-Allyl Proline,or cyclic Glycyl-Alkyl Proline or cyclic Glycyl-2-Methyl-Proline(cPMeG), or a combination thereof, collectively called a cPG compound,to said mammal; wherein said mammal is treated for symptoms of Dementiawith Lewy bodies.